© 1995 American Heart Association, Inc.
Articles |
Reduction of Intimal Hyperplasia by Naroparcil, a 4-Methylumbelliferyl ß-D-Xyloside Analogue, After Arterial Injury in the Hypercholesterolemic Rabbit
From the Unité Physiopathologie du Coeur et des Artères (P.G.S., O.T.), Faculté Xavier Bichat, Paris, France; Laboratoires Fournier-Dijon (C.R., D.P., P.M., P. Bélichard), Dijon, France; and INSERM U28, Pathologie Rénale et Vasculaire (M.Z., P. Bruneval), Hôpital Broussais, Paris, France.
Correspondence to Philippe Gabriel Steg, MD, Service de Cardiologie, Hôpital Bichat, 46 rue H. Huchard, 75018 Paris, France.
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Abstract |
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Abstract 4-Methylumbelliferyl ß-D-xylosides (ß-D-xylosides) inhibit proteoglycan synthesis, and this is associated with reduced proliferation and extracellular matrix production by vascular smooth muscle cells. This study evaluated whether treatment with naroparcil, a ß-D-xyloside analogue, reduced intimal hyperplasia after arterial injury in the hypercholesterolemic rabbit. Forty-two rabbits were assigned to three groups that received either a 1% cholesterol-enriched diet (group 1, n=15) or the same diet with added 100 mg · kg-1 naroparcil (group 2, n=15) or 300 mg · kg-1 naroparcil (group 3, n=12). All animals underwent iliac artery endothelial abrasion at day 14 and were killed at day 56. At the time of death, the angiographic minimal luminal diameter was significantly larger in both treated groups. Morphometric analysis showed a larger luminal area in treated rabbits (groups 2 and 3) compared with control rabbits (group 1) (0.75±0.54 and 0.85±0.61 mm2 versus 0.32±0.25 mm2, respectively; P<.05), with a decreased intimal thickness in groups 2 and 3 (average reduction of 37% and 39%, respectively, compared with group 1; P<.05) but without changes in medial area. Total vessel area was comparable among all groups. In the media, immunohistochemistry suggested reduced infiltration by macrophages and a larger fractional area of smooth muscle cells. There were no differences in plasma or arterial wall cholesterol content between groups. Plasma levels of glycosaminoglycans and dermatan sulfate content were increased only in group 3. In this model, oral treatment with naroparcil appears to preserve the arterial lumen and reduce intimal thickness after arterial injury.
Key Words: angioplasty • restenosis • arterial injury • glycosaminoglycans • proteoglycans
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Introduction |
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Intimal hyperplasia plays an important role in the response to arterial injury (such as that observed in restenosis after angioplasty)1 as well as in the early lesions of atherosclerosis.2 Intimal hyperplasia is due to smooth muscle cell migration from the media to the intima and subsequent proliferation, but it is also due to the exaggerated production of extracellular matrix by smooth muscle cells.3 Indeed, it has been shown that in volume, extracellular matrix is the major component of the intimal lesions induced by arterial injury4 5 and may play a more important role in the genesis of restenosis than actual cell proliferation.6 Arterial proteoglycans have an important role in extracellular matrix assembly, in cell-cell and cell-matrix adhesion, and in the regulation of cell growth and differentiation.7 There is evidence that they accumulate in early phases of atherogenesis and may participate in the genesis of complications.
ß-D-Xylosides are able to specifically inhibit proteoglycan synthesis by interfering with the galactosyl transferase system, which initiates GAG chain synthesis.8 These molecules compete with the cell’s endogenous core proteins for the synthesis of xylosilated GAG chains. These chondroitin sulfate, dermatan sulfate, or heparan sulfate chains are rapidly secreted from the cell into the bloodstream.9 It has been shown in cell culture that this results in inhibition of proliferation and extracellular matrix production by rodent and human vascular smooth muscle cells.7 10 11 In addition, ß-D-xylosides can prevent smooth muscle cell proliferation by proteoglycan-independent mechanisms.10 11 12 Finally, there may be potentially negative structural effects produced by interference with GAG synthesis, as shown in other organs where ß-D-xylosides inhibit morphogenesis and modify protein synthesis.13
The aims of the present study were to evaluate whether an orally active ß-D-xyloside analogue, naroparcil([4-(4-cyanobenzoyl)-phenyl]-1,5-dithio-ß-D-xylopyranoside), reduced intimal hyperplasia induced by arterial injury in the hypercholesterolemic rabbit and to study the subsequent changes in cellular components of the intima and media.
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Materials and Methods |
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Study Design
This was a randomized controlled study of the prevention by naroparcil of intimal hyperplasia induced by iliac arterial abrasion in New Zealand White rabbits fed an atherogenic diet. The present study was performed in accordance with the institutional guidelines for animal research. Rabbits were randomly allocated to three groups that received either a fixed amount (120 g) of a standard atherogenic diet containing 1% cholesterol (Aliment C15, Piètrement) (group 1, n=15) or the same diet containing a low dose (100 mg · kg-1 naroparcil per 100 g diet; group 2, n=15) or a high dose of naroparcil (300 mg · kg-1 naroparcil per 100 g diet; group 3, n=12). The diet and treatments were begun 14 days before arterial injury (day 14) and were maintained until death 42 days later (day 56). The high dose was selected from preliminary data suggesting both efficacy in the prevention of intimal hyperplasia in the rabbit and increased levels of circulating GAGs such as dermatan sulfate, whereas the low dose was selected because it does not lead to increased plasma levels of dermatan sulfate in the rabbit (authors’ unpublished data, 1994).
Housing and Feeding
New Zealand White rabbits weighing 3 kg (2.5 to 3.7 kg) received
120 g of a 1% cholesterol-enriched diet daily that
contained no naroparcil (group 1), 100 mg · kg-1
naroparcil (group 2), or 300 mg · kg-1 naroparcil
(group 3). Food intake was measured daily and showed an average daily
naroparcil intake of 82±8 mg · kg-1 body wt in group
2 and 262±23 mg · kg-1 body wt in group 3. Since a
pilot study had indicated a lowered food intake during the 48 hours
after balloon injury, groups 2 and 3 animals received half their daily
drug dosage dissolved in 20 mL BID of water directly through a gastric
tube during that period. Food intake was lowered to a standard dose of
60 g for the same period in all groups.
Balloon Injury
Anesthesia was induced by acepromazine (0.7
mg · kg-1 IM) and intravenous
pentobarbital and maintained by intermittent intravenous
injections of the latter (total dose, 6 to 10
mg · kg-1). The femoral artery was surgically exposed,
and a 4F latex balloon catheter (Baxter) was inserted and advanced
retrogradely into the aorta under fluoroscopic guidance. After
intra-arterial injection of 200 µg of isosorbide
dinitrate, an angiogram was recorded after placement of a
calibrated radiopaque grid (Namic Medical Systems) at the level of the
aorta. The contrast agent used was amidodiatrizoate (Schering).
Fluoroscopy and angiography were performed by using a CGR Optascop
x-ray tube. The latex balloon was then inflated with 1.5 mL of
saline and withdrawn five times through the entire iliac artery. This
operation was repeated in the contralateral iliac artery.
Follow-up and Preservation of Samples
Six weeks after arterial injury,
anesthesia was induced in a similar fashion, and a midline
laparotomy was performed, allowing surgical exposure of the abdominal
aorta. A 6F arterial sheath was then inserted just below
the level of the renal arteries, and after injection of 200 µg of
isosorbide dinitrate, a follow-up angiogram was recorded. After
ligature and excision of the left iliac artery, which was
snap-frozen in liquid nitrogen for subsequent biochemical
analysis, rabbits were given a lethal dose of sodium
pentobarbital. The right iliac artery was perfusion-fixed with
3.7% buffered formalin at 100 mm Hg for 15 minutes through the
arterial sheath and with efflux through the
inferior vena cava to maintain the artery at in vivo
dimensions. It was then excised and placed in 3.7% buffered formalin
for 48 hours before processing.
Quantitative Angiographic Analysis
All basal and follow-up angiograms were analyzed in
blind fashion by two independent observers using the SAMBA
2005 image analysis system (version 3.02, Alcatel TITN) with a
512'512 eight-bit matrix driven by a personal computer. The
angiographic films were used for acquisitions via a 28-mm f/2.8 Nikkor
lens connected to a mono CCD camera (XC77-CE, Sony). Digitized
angiographic images were then processed by a custom-made software
that was designed to provide the minimal absolute luminal diameters of
each vessel, with the 10-mm grid used for internal pixel calibration.
When an arterial segment was occluded, the minimal diameter
of that vessel was considered to be 0. The interobserver correlation
coefficients of angiographic readings were .99 for both baseline and
follow-up angiograms. All absolute diameter values differing by
either 0.3 mm or 30% between observers were redone by consensus, and
the second reading was used for analysis.
Tissue Preparation
Five samples of 2 mm were systematically taken over the entire
length of each right iliac artery. After dehydration in alcohol and
xylene, samples were embedded in paraffin, and serial 5-µm cross
sections were performed and placed on
poly-L-lysine-coated slides (Sigma Chemical Co). These
sections were used for qualitative and quantitative histomorphometric
analysis.
Histochemical and Immunohistochemical Techniques
Paraffin sections were deparaffinized and rehydrated.
Thereafter, they were stained with orcein for elastin
characterization.14 Serial sections from each right iliac
artery were immunohistochemically labeled with RAM-11 (Dako), a
monoclonal antibody specifically targeted against the cytoplasm of
rabbit macrophages, and with HHF-35 (Enzo Biochemical), a
monoclonal antibody for the 
and 
isotypes of actin. These
antibodies have been previously used to study, respectively,
macrophage-predominant and smooth muscle
cell-predominant regions from intimal lesions in the
rabbit.15 16 Sections were also incubated with 
di-6S
(ICN Immunobiologicals), an antibody that recognizes 6-sulfated
chondroitin sulfate proteoglycans after chondroitinase ABC (Sigma)
pretreatment (2-hour incubation of the sections with 1.5 U/mL of
chondroitinase ABC in Tris buffer at 37°C, 0.05 mol/L, and pH
8).17 This antibody has previously been used to study and
quantify the distribution of chondroitin sulfate proteoglycan in the
intact and deendothelialized wall of rabbit
arteries.18 19 For each antibody, the
streptavidin-biotin peroxidase technique with diaminobenzidine was
applied. No counterstaining was performed. Negative controls were
obtained by omission of the primary antibody (HHF-35 and RAM-11) and by
omission of the chondroitinase ABC digestion for chondroitin sulfate
(di-6S) labeling.
Quantitative Morphometric Analysis
Computer-assisted morphometric analysis was
performed with the SAMBA 2005 image analysis system
(Alcatel TITN). Briefly, each of the five orcein-stained cross
sections was digitized via an Olympus microscope (Optiphot-2) connected
to a tri-CCD color camera (XC-007P, Sony) and to the SAMBA
image analysis system. Image acquisition was performed through
red, green, and blue channels with a 512x512 eight-bit matrix.
Each pixel was assigned a gray value ranging from 0 to 255 in each
channel. Standardization of the light intensity was achieved by
application of a pseudocolor mode before each measurement. The computer
segmentation of the positively stained areas was based on the range
threshold of the red-green-blue-digitized image. The lower
threshold value was measured in the nonstained parts of the
histochemically stained cross sections and in the control cross
sections for immunohistochemically-stained specimens. The upper
threshold value was evaluated from red blood cells that highly express
peroxidase activity.
Histochemistry
Morphometric analysis of all sections was performed by
using custom-made programs, as previously described.20
The entire vessel circumference was displayed at x64 final
magnification. Boundaries between adventitia, media, and intima were
automatically detected, traced, and displayed for the operator, who
could manually correct the separation between structures if necessary.
Analysis was performed on tissue sections stained by orcein,
which produces intense staining of the internal and external elastic
laminae. Vessels with extensive dissections precluding adequate
qualitative analysis of the intimal and medial boundaries were
excluded from the analysis (n=4, all in group 1). Luminal,
intimal, and medial areas, internal and external elastic laminar
perimeters, and intimal and medial thicknesses (mean and maximal) were
then calculated after internal calibration of pixel size. The
"external elastic lamina-enclosed area" (lumen+intima+media)
as well as the "residual lumen index" (luminal
area/luminal+intimal area) were also provided. All quantitative
histological measurements reported were made at the
point of smallest minimal luminal diameter on morphometric
analysis.
Immunohistochemistry
Custom-made software was developed for each
immunohistochemical labeling method to detect and measure the labeled
areas in each cross section, in the intima as well as in the media, at
a magnification of x122. Measurements were performed over the entire
cross section of each artery, and results were expressed as fractional
areas of intima and media predominantly labeled by RAM-11 (Fig 1
), HHF-35 (Fig 2), and di-6S
antibodies. The mean immunostained fractional areas were
calculated for each artery. Control slides were used for the
determination of nonspecific background. The reproducibility of the
analysis was controlled by comparison of analyses of
elementary field measures through various lenses and by iterative
measurements, as well as by analysis of various sections on
different slides obtained from the same tissue block. This quantitative
method of analysis has been used and validated
previously.21 22
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Biochemistry
Drug Dosages
Plasma measurements of LF 90054
([4-(4-cyanobenzylhydroxy)-phenyl]-1.5-dithio-ß-D-xylopyranoside),
the circulating form of naroparcil, were performed at day 34 of
treatment (on average, 2 and 23 hours after food intake) by using a
solid-phase extraction on C-18 cartridges (Analytichem). After
reverse-phase chromatography on a C-18 column,
detection was made from UV absorbance.
Serum Cholesterol
Fasting blood samples were obtained the day before and 2, 5, and
8 weeks after beginning the atherogenic diet. Total serum
cholesterol was determined enzymatically on a Cobas
analyzer (Cobas-Fara, Hoffmann La Roche) with the
Biomérieux total cholesterol reagent (PAP kit,
Biomérieux).
Arterial Wall Cholesterol
The adventitia of the left iliac artery was stripped, and the
remaining intima and media were minced. Lipids were extracted in
ether/ethanol (1:2) by using the procedure described by Smith and
Slater.23 The extracts were redissolved in
chloroform/isopropanol (2:)1). Cholesterol was then
extracted with heptane and measured by gas-phase
chromatography with a Varian Star 3400
apparatus.
Plasma GAGs
Arterial blood samples (20 mL) were collected on
citrate (1 vol of 3.8% [wt/vol] sodium citrate for 9 vol of blood)
immediately before the animals were killed. After
centrifugation (2400g, 20 minutes, and
20°C), the platelet-poor plasma was kept frozen (-70°C)
for further analysis. Samples were then pooled in each group.
Proteolytic digestion was performed by incubating plasma with pronase E
type XIV for 48 hours at 50°C. Cold trichloroacetic acid was then
added, and samples were kept overnight at 4°C. The supernatant was
recovered by centrifugation and dialyzed (Spectrapor 3,
Polylabo). The resulting supernatant was then precipitated by addition
of CPC (final concentration, 0.1%). After dissolution of GAG-CPC
complexes in 2 mol/L NaCl, GAGs were precipitated by the addition of 5
vol of 95% (vol/vol) ethanol and recovered by
centrifugation (15 minutes, 1500g, and
4°C), dried under vacuum, and dissolved in 0.9% NaCl. Aliquots were
then desalted on PD10 columns (Pharmacia-LKB Biotechnology) and
lyophilized. The UA content of the final GAG extract was determined on
lyophilized samples by using the methods of Bitter and
Muir.24 Analysis of disaccharides
produced by enzymatic digestion of purified GAG was then performed by
high-performance liquid chromatography
(Dionex Bio L.C) using the techniques of Linhardt et
al.25
Statistics
Data were expressed as mean±SD. Unless otherwise specified,
comparisons between groups for continuous variables were performed
by ANOVA. When significant, post hoc comparisons between groups were
performed by Scheffé’s method. A value of P<.05 was
considered significant. Comparisons between groups for categorical
variables used the 
2 method. Statistics were
computed by using STATVIEW II software (Abacus
Concepts).
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Results |
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Weight and Food Intake
There was no significant difference between groups in food intake along the entire treatment period. Similarly, the weight of the animals was comparable between groups at baseline, at the time of arterial injury, and at the time of death (one-way ANOVA, Table 1
).
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Serum Cholesterol
In all the groups, the cholesterol-enriched diet
induced a progressive increase in total serum cholesterol.
However, there was no significant difference between groups in
cholesterol at any time point (two-way ANOVA, Table 2).
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Angiographic Analysis
The minimal luminal diameters did not differ significantly between
groups at the time of arterial injury (day 14), although
there was a nonsignificant trend toward a larger minimal luminal
diameter in both treated groups. At the time of death (day 56, ie, 42
days after arterial injury), there was a significantly
larger minimal luminal diameter in both treated groups than in the
control group (Fig 3). The number of occluded iliac
arteries were five, zero, and two in groups 1, 2, and 3, respectively
(P=NS by 2 analysis).
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Qualitative Histopathological Analysis
All the arteries showed evidence of intimal thickening, mostly
eccentric and composed of an abundant extracellular matrix, smooth
muscle cells, and foamy macrophages (Fig 4).
There was constant focal disruption or double outlining of the internal
elastic lamina and varying degrees of thickening and infiltration of
the media by macrophages.
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Quantitative Histomorphometry
Results from the quantitative morphometric analysis are
displayed in Fig 5. There was a significantly larger
luminal area at the time of death in both treated groups than in the
control group (133% and 165% increases from group 1 values in groups
2 and 3, respectively; P<.05 for both). The mean intimal
thickness was significantly lower in both treated groups than in the
control group (37% and 39% reduction from group 1 values for groups 2
and 3, respectively; P<.05 for both). In addition, there
was a reduction of intimal area in the treated groups (30% and 27% in
groups 2 and 3, respectively; P<.01 and P<.02,
respectively, versus group 1), whereas the medial areas (as well as the
external elastic lamina-enclosed area) were comparable between
groups. Similarly, the residual lumen index was significantly lower in
the control group than in the treated groups (146% and 154%
increases, respectively; P<.05 for both).
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Immunohistochemical Morphometric Analysis
The morphometric analysis (Fig 6) of the
fractional area of components showed, in the intima, a nonsignificant
trend toward a decrease in the percentage of RAM-11-immunolabeled areas
(relative decreases of 19% and 10% in groups 2 and 3, respectively;
P=NS) and an increase in HHF-35-immunostained
areas in both treated groups (relative increases of 48% and 28% in
groups 2 and 3, respectively; P=NS). In the media,
RAM-11-immunolabeled areas were significantly lower in groups 2 and 3
than in the control group (relative decreases of 45% and 46%,
respectively; P<.05 for both) with a significantly higher
HHF-35-immunostained fractional area (relative increases of
40% and 83%, respectively; P<.05 for both). For both the
intima and the media, the fractional areas labeled by di-6S were not
significantly different between groups.
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Arterial Wall Lipid Content
The total cholesterol content of the iliac
arterial segments analyzed was comparable in all
three groups (107±5 versus 107±10 and 107±8
mg · g-1 of dry defated tissue for groups 1, 2, and 3,
respectively).
Plasma GAGs
At the time of death, pooled plasma GAG levels were unchanged in
group 2 versus group 1 (3.9 versus 3.4 µg
UA · mL-1, respectively); they amounted to 8.2
µg UA · mL-1 in group 3. In addition, 6-sulfated
chondroitin sulfate GAGs in the plasma represented 2.7%,
21%, and 28.2% of total GAGs in groups 1, 2, and 3, respectively. The
dermatan sulfate content was undetectable in groups 1 and 2 but reached
9.1% of total GAGs in group 3.
Drug Dosages
The plasma concentrations of LF 90054 were 2074±396 and 1831±930
ng · mL-1 in group 2 at 2 and 23 hours, respectively,
whereas they were 5793±2275 and 5958±1956 ng · mL-1
in group 3 (P<.001 for both comparisons with group 2).
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Discussion |
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The synthetic ß-D-xyloside naroparcil was first described as an orally active venous antithrombotic agent that promotes heparin cofactor II-thrombin complex formation, probably by increasing circulating dermatan sulfate-like GAG levels.9 26 ß-D-Xylosides act as primers for GAG chain initiation and compete with the xylosilated core protein at the level of the second glycosyltransferase, which adds galactose to a xylose residue on the core protein. The composition of the GAGs induced by ß-D-xylosides depends on the structure of the aglycone part of this compound.27 These GAGs are secreted from the cell.
Vascular smooth muscle cells are the main producers of proteoglycans in the arterial wall.28 The role of vascular proteoglycans in the pathogenesis of vascular lesions leading to atherosclerosis and restenosis after angioplasty has been emphasized previously.5 29 A coincident increase in proteoglycan synthesis and cellular proliferation has been demonstrated in a variety of tissues.11 Therefore, interference with proteoglycan metabolism may be expected to modulate proliferation, and indeed, ß-D-xylosides inhibit cell proliferation10 11 30 and, in particular, arterial smooth muscle cell proliferation in vitro. Thus, we examined the effects of naroparcil on intimal hyperplasia after balloon iliac injury in the hypercholesterolemic rabbit.
Quantitative angiographic and histomorphometric analyses showed that continued oral treatment with naroparcil resulted in preservation of the luminal area and decreased intimal thickness and area 42 days after arterial injury. The data do not demonstrate a clear dose-response relation between groups 2 and 3, suggesting that the effect of naroparcil may be generic rather than specific. Considering recent reports suggesting arterial remodeling in response to injury,31 32 33 34 35 it is noteworthy that treatment with naroparcil did not affect global vessel size, measured by the external elastic lamina-enclosed area, suggesting that the differences between groups cannot be ascribed to differences in remodeling. However, since a control nondilated segment was not measured for reference, whether remodeling occurs in the present study remains unclear. The qualitative histological analysis of the components of the vessel wall showed no change in the fractional areas of each of the components of the intima but an increase of the fractional area of HHF-35-stained cells and a decrease in that of RAM-11-stained cells in the media. There was no difference between naroparcil-treated and control animals with respect to the 6-sulfated chondroitin sulfate chains in the intima or in the media.
The mechanism by which naroparcil reduced intimal hyperplasia in this model remains unclear. There was a reduction of macrophage infiltration in the media, and it has been shown that macrophages contribute significantly to the cellular response to injury in the present experimental model, in which a high-cholesterol diet favors macrophage invasion and foam cell formation.15 36 37 Since there was no difference in arterial wall cholesterol content or in total plasma cholesterol between groups, this reduced macrophage infiltration cannot be directly ascribed to changes in plasma or arterial cholesterol. Likewise, it is unlikely that preservation of the angiographic lumen in the treated groups was due solely to a vasomotor effect, since angiography was performed under maximal vasodilation and since these results were confirmed by histomorphometric analysis, showing reduced intimal thickness and preservation of luminal area in these groups. Since HHF-35 preferentially labels smooth muscle cells with a contractile phenotype,14 38 the increased HHF-35-positive fractional area in the media of naroparcil-treated animals, along with the absence of difference in medial thickness or medial lipid content, suggests a higher percentage of medial cells with a preserved contractile phenotype in the treated groups. Such prevention of phenotypic modulation from a contractile to a synthetic phenotype has been observed in cultured rat aortic smooth muscle cells treated with ß-D-xyloside10 and in smooth muscle cells of balloon-denuded carotid arteries from heparin-treated rats.39 This phenomenon could be a basis for the reduced intimal thickness in naroparcil-treated animals, since phenotypic modulation is a key event in the sequence, leading to migration, proliferation of smooth muscle cells, and increased extracellular matrix production.3
On the other hand, the mechanism by which naroparcil beneficially acts
on intimal hyperplasia may also be independent from the inhibition of
proteoglycan synthesis. Proliferation of several cell types can be
inhibited by ß- and also by -xylosides, which do not interfere
with proteoglycan synthesis.10 11 12 Moreover, it has
recently been shown that both - and ß-xylosides inhibit the
synthesis of glycolipids that are known to have marked effects on
cellular proliferation.40 However, it must be emphasized
that in the present study, cell proliferation was not measured,
precluding definitive conclusions about the mechanism of naroparcil
benefit.
Although the mechanism by which naroparcil limits intimal hyperplasia may be proteoglycan independent, we examined the effects of naroparcil treatment on arterial wall and plasma proteoglycans. Vascular wall proteoglycans are quite heterogeneous, and their composition varies among animal species. In the rabbit arterial wall, they are mainly a mixture of isomeric chondroitin sulfates (chondroitin sulfate-dermatan sulfate proteoglycans), of which 80% have 6-sulfated chondroitin sulfate chains.41 There was apparently no quantitative difference in the 6-sulfated chondroitin sulfate chain fraction in the injured iliac arterial wall between naroparcil-treated and control animals. However, in the plasma, the concentration of 6-sulfated chondroitin sulfate chains was increased 8 and 23 times in groups 2 and 3, respectively, over that of control animals. In addition, changes in proteoglycan composition of the arterial wall at earlier time points may have been missed, since the only analysis of arterial wall proteoglycans was at the time of autopsy.
As previously stated, naroparcil has been shown, in a rabbit venous thrombosis model, to possess antithrombotic properties thought to be mediated via an increase in dermatan sulfate-like GAGs of the same magnitude as that reported in the present study. In addition, in vivo studies have demonstrated an antithrombotic effect of dermatan sulfate.42 43 44 45 46 Thus, since treatment was started 14 days before injury, a naroparcil-induced increase in plasma GAGs (confirmed in the present study) may have participated in the prevention of intimal hyperplasia through its antithrombotic effect. In this respect, the role of early platelet adhesion and local thrombosis in the initiation of intimal hyperplasia has been emphasized previously.47 Nevertheless, a similar degree of prevention of intimal hyperplasia was achieved in both treated groups, whereas circulating dermatan sulfate levels were only detectable in the latter. In addition, the absence of difference between groups in the number of total occlusions further suggests that the results cannot be entirely explained by an antithrombotic effect of naroparcil.
Despite the limits of experimental models, particularly of the rabbit model, for the study of atherosclerosis or restenosis after angioplasty, the rabbit iliac injury model has several advantages: severely stenotic lesions develop within weeks (average diameter of stenosis, 75±18% in the present study; data not shown), and the intimal lesions are made of both smooth muscle cells and extracellular matrix, which is akin to the early lesions of atherosclerosis or of restenosis. In addition, rabbit arteries have the proteoglycan composition closest to that of humans.29 However, several differences with restenosis after angioplasty must be pointed out and should induce caution before extrapolating those results to other species. The lesions induced by diet and injury in the rabbit are rich in foam cells and are highly infiltrated by both intracellular and extracellular lipids.37 In addition, although injury was performed in vessels already infiltrated by activated macrophages,48 the vessels were not truly atherosclerotic, and the extent of injury produced by the latex balloon catheter differed from that created in the clinical situation of angioplasty.
In summary, in the hypercholesterolemic rabbit injury model, oral treatment with naroparcil, a ß-D-xyloside analogue, resulted in preservation of the arterial lumen on both quantitative angiographic and histomorphometric analyses, with a reduced intimal thickness. This effect was not related to decreased plasma or arterial wall cholesterol. Whether it was directly linked to interference with proteoglycan synthesis, to a direct antiproliferative effect of the synthesized proteoglycans, or to some other effect on cell metabolism has not yet been elucidated. However, a preserved contractile phenotype of medial smooth muscle cells and a decreased macrophage invasion of the media may participate in the reduction of intimal hyperplasia by naroparcil. This compound deserves further evaluation as a candidate therapy for restenosis after angioplasty.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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We are indebted to André Dubois for statistical analysis, to Dominique Coup for GAG analysis, and to Marie-France Bélair for technical assistance.
Received January 23, 1995; accepted July 5, 1995.
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References |
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1. Strauss BH, Chisholm RJ, Keeley FW, Gotlieb AI, Logan RA, Armstrong PW. Extracellular matrix remodeling after balloon angioplasty injury in a rabbit model of restenosis. Circ Res.. 1994;75:650-658.
2. Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990’s. Nature.. 1993;362:801-809. [Medline] [Order article via Infotrieve]
3. Clowes AW, Reidy MA, Clowes MM. Mechanisms of stenosis after arterial injury. Lab Invest.. 1983;49:208-215. [Medline] [Order article via Infotrieve]
4. Forrester JS, Fishbein M, Helfant R, Fagin J. A paradigm for restenosis based on cell biology: clues for the development of new preventive therapies. J Am Coll Cardiol.. 1991;17:758-769. [Abstract]
5. Riessen R, Isner JM, Blessing E, Nikol S, Wight TN. Characterization of the extracellular matrix of coronary restenosis lesions: distribution of the extracellular matrix proteoglycans biglycan and decorin. Am J Pathol.. 1994;144:962-974. [Abstract]
6.
O’Brien ER, Alpers CE, Stewart DK, Ferguson M, Tran
N, Gordon D, Benditt EP, Hinohara T, Simpson JB, Schwartz SM.
Proliferation in primary and restenotic
coronary atherectomy tissue: implications for antiproliferative
therapy. Circ Res.. 1993;73:223-231.
7. Wight TN, Potter-Perigo S, Aulinskas T. Proteoglycans and vascular cell proliferation. Am Rev Respir Dis.. 1989;140:1132-1135. [Medline] [Order article via Infotrieve]
8.
Schwartz NB, Galligani L, Ho PL, Dorfman A.
Stimulation of synthesis of free chondroitin sulfate chains by
ß-D-xylosides in cultured cells. Proc Natl
Acad Sci U S A.. 1974;71:4047-4051.
9.
Masson P, Coup D, Millet J, Brown NL. The
effect of the ß-D-xyloside naroparcil on circulating
plasma glycosaminoglycans: an explanation for its
known antithrombotic activity in the rabbit. J
Biol Chem.. 1995;270:2662-2668.
10.
Hamati BF, Britton EL, Carey DJ. Inhibition of
proteoglycan synthesis alters extracellular matrix deposition,
proliferation, and cytoskeletal organization of rat aortic smooth
muscle cells in culture. J Cell Biol.. 1989;108:2495-2505.
11. Potter-Perigo S, Braun KR, Schönherr E, Wight TN. Altered proteoglycan synthesis via the false acceptor pathway can be dissociated from ß-D-xyloside inhibition of proliferation. Arch Biochem Biophys.. 1992;297:101-109. [Medline] [Order article via Infotrieve]
12. Kolset SO, Sakurai K, Ivked I, Øvervatn A, Suzuki S. The effects of ß-D-xylosides on the proliferation and proteoglycan biosynthesis of monoblastic U-937 cells. Biochem J.. 1990;265:637-645. [Medline] [Order article via Infotrieve]
13.
Canfield AE, Sutton AB, Hiscock DRR, Gallagher JT,
Schor AM. and ß-xylosides modulate the synthesis of
fibronectin and thrombospondin-1 by endothelial
cells. Biochim Biophys Acta.. 1994;1200:249-258. [Medline]
[Order article via Infotrieve]
14. Ganter P, Jollès G. Histochimie Normale et Pathologique. Paris, France: Gauthier Villars; 1969.
15. Tsukada T, Rosenfeld M, Ross R, Gown AM. Immunocytochemical analysis of cellular components in atherosclerotic lesions. Arteriosclerosis.. 1986;6:601-613. [Abstract]
16.
Stadius ML, Gown AM, Kernoff R, Collins CL. Cell
proliferation after balloon injury of iliac arteries in the
cholesterol-fed New Zealand White rabbit.
Arterioscler Thromb.. 1994;14:727-733.
17. Couchman JR, Caterson B, Christner JE, Backer JR. Mapping by monoclonal antibody detection of glycosaminoglycans in connective tissues. Nature.. 1984;307:650-652. [Medline] [Order article via Infotrieve]
18. Srinivasan SR, Xu J, Vijayagopal P, Rhadakrishnamurthy B, Berenson GS. Injury to the arterial wall of rabbits produces proteoglycan variants with enhanced low-density lipoprotein-binding property. Biochem Biophys Acta.. 1993;1168:158-166. [Medline] [Order article via Infotrieve]
19. Galis ZS, Alavi MZ, Moore S. Co-localization of aortic apolipoprotein B and chondroitin sulfate in an injury model of atherosclerosis. Am J Pathol.. 1993;142:1432-1438. [Abstract]
20. Pruneau D, Luccarini JM, Robert C, Bélichard P. Induction of kinin B1 receptor-dependent vasoconstriction following balloon catheter injury to the rabbit carotid artery. Br J Pharmacol.. 1994;111:1029-1034. [Medline] [Order article via Infotrieve]
21. Schmid KW, Morgan JM, Öfner D, Hittmair A, Haywood S, Jaszani B. Quantitative immunohistochemical evaluation by image analysis of metallothionein in copper-loaded rat kidney. J Histochem Cytochem.. 1993;41:727-731. [Abstract]
22.
Qiao Y, Yokoyama M, Kameyama K, Asano G. Effects
of vitamin E on vascular integrity in cholesterol-fed
guinea pigs. Arterioscler Thromb.. 1993;13:1885-1892.
23. Smith EB, Slater RS. The microdissection of large atherosclerotic plaques to give morphologically and topographically defined fractions for analysis. Atherosclerosis.. 1972;15:37-56. [Medline] [Order article via Infotrieve]
24. Bitter T, Muir HM. A modified uronic acid carbazol reaction. Anal Biochem.. 1962;4:330-334. [Medline] [Order article via Infotrieve]
25. Linhardt RJ, Gu KN, Lopanathan D, Carter SR. Analysis of glycosaminglycan-derived oligosaccharides using reversed phase ion-pairing and ion exchange chromatography with suppressed conductivity detection. Anal Biochem.. 1985;181:288-296.
26. Millet J, Theveniaux J, Brown NL. The venous antithrombotic profile of naroparcil in the rabbit. Thromb Haemost.. 1994;72:874-879. [Medline] [Order article via Infotrieve]
27.
Fritz TA, Lugemwa FN, Sarkar AK, Esko JD.
Biosynthesis of heparan sulfate on ß-D-xylosides
depends on aglycone structure. J Biol
Chem.. 1994;269:300-307.
28. Radhakrishnamurthy B, Srinivasan SR, Viajayagopal P, Berenson GS. Arterial wall proteoglycans: biological properties related to pathogenesis of atherosclerosis. Eur Heart J. 1990;11(suppl E):148-157.
29.
Wight TN. Cell biology of arterial
proteoglycans. Arteriosclerosis.. 1989;9:1-20.
30.
Johnston LS, Keller JM. The effect of
ß-D-xylosides on heparan sulfate synthesis by
SV40-transformed Swiss mouse 3T3 cells. J Biol
Chem.. 1979;254:2575-2578.
31.
Post M, Borst C, Kuntz RE. The relative
importance of arterial remodeling compared with intimal
hyperplasia in lumen renarrowing after balloon angioplasty: a study in
the normal rabbit and the hypercholesterolemic
Yucatan micropig. Circulation. 1994;89:2816-2821.
32.
Kakuta T, Currier JW, Haudenschild CC, Ryan TJ, Faxon
DP. Differences in compensatory vessel enlargement, not intimal
formation, account for restenosis after angioplasty in the
hypercholesterolemic rabbit model.
Circulation. 1994;89:2809-2815.
33.
Lafont A, Guzman LA, Whitlow PL, Goormastic M, Cornhill
JF, Chisolm G. Restenosis after experimental
angioplasty: intimal, medial, and adventitial changes associated with
constrictive remodeling. Circ Res.. 1995;76:996-1002.
34.
Glagov S. Intimal hyperplasia, vascular
modeling, and the restenosis problem.
Circulation. 1994;89:2888-2891.
35.
Isner JM. Vascular remodeling.
Circulation. 1994;89:2937-2941.
36.
Hanke H, Strohschneider T, Oberhoff M, Betz E, Karsch
KR. Time course of smooth muscle cell proliferation in the
intima and media of arteries following experimental
angioplasty. Circ Res.. 1990;67:651-659.
37.
Weidinger FF, McLenachan JM, Cybulsky MI, Fallon JT,
Hollenberg NK, Cooke JP, Ganz P.
Hypercholesterolemia enhances
macrophage recruitment and dysfunction of regenerated
endothelium after balloon injury of the rabbit iliac
artery. Circulation. 1991;84:755-767.
38. Wanibuchi H, Dingemans KP, Becker AE, Ueda M, Naruko T, Tanizawa S, Nakamura K. Is the Watanabe heritable hyperlipidemic rabbit a suitable experimental model for percutaneous transluminal coronary angioplasty in humans?: a light microscopic, immunohistochemical and ultrastructural study. J Am Coll Cardiol.. 1993;21:1490-1496. [Abstract]
39.
Clowes AM, Clowes MM, Kocher O, Ropraz P, Charponnier
C, Gabbiani G. Arterial smooth muscle cells in vivo:
relationship between actin isoform expression and mitogenesis and their
modulation by heparin. J Cell Biol.. 1988;107:1939-1945.
40.
Freeze HH, Sampath D, Varki A. - and
ß-xylosides alter glycolipid synthesis in human melanoma and
Chinese hamster ovary cells. J Biol Chem.. 1993;268:1618-1627.
41. Radhakrishnamurthy B, Srinivasan SR, Ruiz HA, Dalferes ER, Berenson GS. Variations in the composition of arterial wall isomeric chondroitin sulfate proteoglycans among different animal species. Comp Biochem Physiol [B].. 1990;97:355-362. [Medline] [Order article via Infotrieve]
42. Linhardt RJ, Desai UR, Liu J, Pervin A, Hoppensteadt D, Fareed J. Low molecular weight dermatan sulfate as an anti-thrombotic agent: structure activity relationship studies. Biochem Pharmacol.. 1994;47:1241-1252. [Medline] [Order article via Infotrieve]
43.
Cadroy Y, Hanson SR, Harker LA. Dermatan sulfate
inhibition of fibrin-rich thrombus formation in nonhuman
primates. Arterioscler Thromb.. 1993;13:1213-1217.
44. Carrié D, Caranobe C, Gabaig AM, Larroche M, Boneu B. Effects of heparin, dermatan sulfate and of their association on the inhibition of venous thrombosis growth in the rabbit. Thromb Haemost.. 1992;68:637-641.[Medline] [Order article via Infotrieve]
45. Gianese F, Lucchelli PE. A survey of the clinical experience with dermatan sulfate. Semin Thromb Hemost.. 1991;17:199-204.
46. Pescador R, Porta R, Mantovani M, Prino G, Casu B, Naggi A, Torri G, Walenga JM, Hoppensteadt DA, Fareed J. Pharmacologic profile of sulfamino-galactosaminoglycans. Semin Thromb Hemost.. 1991;17:74-79.
47. Ip JH, Fuster V, Israel D, Badimon L, Badimon J, Chesebro JH. The role of platelets, thrombin and hyperplasia in restenosis after coronary angioplasty. J Am Coll Cardiol.. 1991;17:77B-88B.
48. Libby P, Schwartz D, Brogi E, Tanaka H, Clinton SK. A cascade model for restenosis: a special case of atherosclerosis progression. Circulation. 1992;86(suppl III):III-47-III-52.
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