115
Glycosaminoglycan Fractions From Human
Arteries Presenting Diverse Susceptibilities
to Atherosclerosis Have Different
Binding Affinities to Plasma LDL
Luiz E.M. Cardoso, Paulo A.S. Mourao
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Abstract The topographic distribution of atherosclerotic
lesions is influenced by biochemical factors intrinsic to the
arterial wall. In the present work we have investigated whether
the composition/chemical structure of glycosaminoglycans
constitutes one of these factors. Normal human arteries were
obtained at necropsy, and in order of decreasing susceptibility
to atherosclerosis, consisted of the abdominal and thoracic
aortas and the iliac and pulmonary arteries. The results
showed similar concentrations of total glycosaminoglycan and
collagen. Of the glycosaminoglycans known to interact with
low-density lipoprotein (LDL), dermatan sulfate was present
in all arteries in comparable concentrations, but the aortas had
a 30% higher content of chondroitin 4/6-sulfate, which in turn
was slightly enriched in 6-sulfated disaccharide units. LDL-
affinity chromatography with dermatan sulfate+chondroitin
4/6-sulfate fractions demonstrated that increasing affinity to
LDL matched an increasing susceptibility to atherosclerosis.
Analysis of glycosaminoglycans in the eluates indicated a
positive correlation between affinity to LDL and increasing
molecular weight and the existence of a fraction of glycosaminoglycans of high affinity to LDL in the aortas only. These
results suggest that arterial glycosaminoglycans participate in
the multifactorial mechanisms that modulate the differential
localization of atherosclerotic lesions. {ArterioscUr Thromb.
1994;1:115-124.)
Key Words • glycosaminoglycans • chondroitin sulfate •
heparan sulfate • atherosclerosis risk factors • LDL •
arterial connective tissue
T
animals have demonstrated that low-density lipoprotein
(LDL) metabolism,11 endothelial permeability to
LDL,12 the glycoprotein coating of endothelial cells,13
and the activity of acid cholesterol esterase14 vary
among vessels, and this variation could be related to
differences in atherosclerosis susceptibility. A further
aspect of arterial constitution that has been correlated
with regional differences in the incidence of atherosclerosis is the degree of folding of the internal elastic
membrane in humans,15 which somehow reflects the
amount of elastin in the vessel wall.
Taken together, the results of these mostly recent
studies show that focal metabolic and biochemical factors inherent in the arterial wall modulate the initiation
and differential localization of atherosclerotic lesions.
Consequently, a precise understanding of the individual
roles of such factors is essential for understanding the
pathogenesis of atherosclerosis.
It is well established that arterial glycosaminoglycans
(GAGs)/proteoglycans play an important role in the
early phases of atherogenesis, since these molecules are
capable of binding and holding plasma LDL that insudates into the arterial wall after endothelial dysfunction. 1618 GAG-LDL complexes are in turn more easily
internalized by macrophages than LDL alone,19 thereby
enhancing the formation of foam cells. Also, GAGs
induce structural alterations to LDL molecules that may
potentiate their atherogenic effects.20 The nature of the
GAG-LDL interaction has therefore been extensively
studied, and it is known that certain GAG species —
including particular populations of a given species —
have greater affinity for LDL.21-24 Thus, should normal
he occurrence of atherosclerotic lesions is associated with a number of risk factors, such as
elevated serum lipid and lipoprotein levels,
hypertension, smoking, sex, and family history.1 However, factors intrinsic to the arterial bed and its bloodcarrying functions also influence the occurrence of
lesions. The primary evidence came from morphological
study of necropsy material, which demonstrated that the
incidence and/or severity of atherosclerotic lesions differed as a function of anatomic location.2-3 Additionally, these studies revealed that in affected arteries,
lesions tended to be located at critical sites, such as the
entrances of vessels and flow dividers, where the effects
of fluid turbulence would be more pronounced. Experimental physiological investigations have supported
these findings68 and have shown that the shear force
due to blood circulation, chiefly at arterial pressures, is
an important cause of endothelial dysfunction at these
sites.9
Still, arterial geometry and pressure are not sufficient
to explain the localization of lesions, since major areas
of high risk for atherosclerosis also occur away from the
ostia and bifurcations10 and some arteries subjected to
normal arterial blood pressure are little affected by
atherosclerosis.3-3 In fact, studies with experimental
Received May 18, 1993; revision accepted October 19, 1993.
From the Departamento de Bioqufmica, Instituto de Cifincias
Biomidicas, Universidade Federal do Rio de Janeiro, Rio de
Janeiro, Brazil.
Correspondence to Luiz E.M. Cardoso, MD, Departamento de
Bioqufmica, Instituto de Cifincias Biom6dicas, Universidade Federal do Rio de Janeiro, Caixa Postal 68041, Rio de Janeiro, RJ,
21944-590, Brazil.
116
Arteriosclerosis and Thrombosis
Vol 14, No 1 January 1994
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variations in GAG composition occur among arteries,
they might lead to variations in LDL retention/alteration capacity in these arteries. Indeed, an early and
succint overview of arterial GAGs indicates that some
variations in composition do seem to exist,25 and similar
anatomic differences have been observed in relation to
the distribution of other components of the extracellular
matrix, such as collagen and elastin,15-26 which intimately associate with GAGs.27-28
Therefore, the purpose of the present study was to
determine whether an eventual anatomic heterogeneity
of arterial GAGs might be a factor contributing to the
differential topographic distribution of atherosclerotic
lesions. To carry out this investigation, our experimental
approach consisted of the following: (1) a more detailed
characterization of GAG composition in normal human
arteries presenting diverse susceptibilities to atherosclerotic lesions; (2) an analysis of the binding affinities of
GAG fractions from these arteries to plasma LDL; and
(3) further characterization of the GAG samples once
their binding affinities had been established.
Methods
Normal human arteries were obtained at necropsy. The
cases consisted of young adults aged 25 to 35 years who had
died accidentally and, so far as could be verified, with no
history of cardiovascular or metabolic diseases. The vessels
comprised the thoracic and abdominal aortas and the iliac and
pulmonary arteries. The iliac artery samples consisted of the
distal segment of the common iliac artery and the proximal
portions of the external and internal iliac arteries. Arterial
segments without macroscopically visible lesions were then
excised, fixed in acetone, and kept at 4°C. After their adventitial layer was stripped off, these segments, which therefore
consisted of the intimal and medial layers, were finely homogenized in acetone and subjected to two changes of 50 volumes
of this solvent for 24 hours each. The final defatted powder
was obtained by drying this material at 60°C. Standard chondroitin 4-sulfate, chondroitin 6-sulfate, dermatan sulfate, and
twice-crystallized papain (15 U/mg protein) were purchased
from Sigma Chemical Co, St Louis, Mo, and cyanogen bromide-activated Sepharose 4B was from Pharmacia, Uppsala,
Sweden. Chondroitin AC lyase (Chondroitinase AC-II) from
Arthrobacter aurescens and chondroitin ABC h/ase from Proteus vulgaris were from Miles Laboratory Co, Elkhart, Ind.
All methods employed for the purification, characterization,
and quantitation of GAGs, as well as the assay for total arterial
collagen, have been described in detail elsewhere.29 Estimations of the concentration of total arterial GAG 30 and collagen31 were made separately for each individual. Subsequent
analyses were made with pooled extracts for each artery.
Isolation of Total Arterial GAGs
One hundred milligrams of the acetone-treated powder was
rehydrated for 24 hours at 4°C in 3.7 mL of 0.1 mol/L sodium
acetate buffer, pH 5.0, containing 5 mmol/L cysteine and 5
mmol/L EDTA. Papain (0.7 mg) was then added to the
mixture, which was incubated at 60°C for 24 hours with mild
agitation. The incubation mixture was centrifuged (2000g, 20
minutes at room temperature), the supernatant was retained,
and the pellet was resuspended in 3.7 mL distilled water and
centrifuged. A 10% cetylpyridinium chloride (CPC) solution
was added to the combined supernatants to a final concentration of 0.5%, and the mixture was left to stand at room
temperature for 24 hours. Next, the solution was centrifuged
and the pellet washed with 15.0 mL of a 0.05% CPC solution.
This pellet, a GAG-CPC complex, was then dissolved with 3.7
mL of a 2-mol/L NaCl/absolute ethanol (100:15, vol/vol)
solution, and the GAGs were precipitated with the addition of
7.4 mL absolute ethanol. After 24 hours at 4°C, the precipitates were collected by centrifugation and washed twice with
15.0 mL 80% ethanol and once with the same volume of
absolute ethanol. The final pellet, which constituted the total
tissue GAG preparation, was dried at 60°C for 30 minutes and
dissolved in 1.1 mL distilled water. Papain digestion under
these conditions completely solubilized all arterial samples,
and the subsequent CPC and ethanol precipitations had a
recovery >90% with regard to a solution containing known
amounts of GAGs.
DEAE-Cellulose Chromatography
Arterial GAGs (100 to 200 mg) were applied to a DEAEcellulose column (2x11 cm, 20-mL bed) equilibrated with 50
mmol/L sodium acetate buffer, pH 6.0. The column was then
washed with 100 mL of this same buffer and subjected to a
linear gradient of 0 to 1.0 mol/L NaCl at a flow rate of 12
mL/h, and fractions of 2 mL were collected. These were
assayed by the carbazole reaction,30 and their salt concentration was estimated by conductivity. Fractions containing
GAGs were pooled and precipitated with three volumes of
absolute ethanol, and the yield was »83% with regard to the
applied material.
Agarose Gel Electrophoresis
GAGs (10 jig) were applied to a 0.5% agarose gel in 0.05
mol/L 1,3-diaminopropane/acetate buffer, pH 9.0. After electrophoresis (120 V for 1 hour), the GAGs in the gel were fixed
with 0.1% N-cetyl-AWAr-trimethylammonium bromide in water, stained with 0.1% toluidine blue in acetic acid/ethanol/
water (0.1:5:5, vol/vol/vol), and washed for about 30 minutes
in acetic acid/ethanol/water (0.1:5:5, vol/vol/vol). The GAGs
in the agarose gel electrophoresis were quantitated by densitometry using a Quick Scan densitometer (Helena Laboratories, Beaumont, Tex).
Polyacrylamide Gel Electrophoresis (PAGE)
The molecular weights of the GAGs were estimated by
PAGE.32-33 GAG samples (10 jig) were applied to a 6%,
1-mm-thick polyacrylamide gel slab and after electrophoresis
(100 V for 30 minutes), the gel was stained with 0.1% toluidine
blue in 1% acetic acid. After staining, the gel was washed for
about 4 hours in 1% acetic acid. The molecular-weight markers were the same as those used previously.23
Enzymatic Degradations
GAGs (100 fig) were incubated with 0.01 U chondroitin
AC lyase, 0.01 U chondroitin ABC lyase,23 or 10 jtL heparitinases34 in 0.05 mol/L ethylenediamine/acetate buffer, pH
8.0, in a final volume of 50 fiL. After incubation at 37°C
PCFC for heparitinases) for 12 hours, the mixtures were
spotted on Whatman No. 1 paper and chromatographed on
isobutyric acid/1 mol/L NH4OH (5:3, vol/vol) for 24 hours.
The products were located by silver nitrate staining and
quantitated by densitometry.
Isolation of LDL From Plasma
LDL (d=1.020 to 1.050 g/mL) was purified by the method of
Havel et al,35 with human plasma obtained from healthy
donors. After sequential ultracentrifugations in potassium
bromide, the LDL preparation was dialyzed exhaustively at
4°C against 0.9% NaCl containing 0.01% EDTA and stored at
4°C. Purity of LDL in this preparation was assessed by agarose
gel electrophoresis in barbital buffer, which showed a single
band. The concentration of LDL in these preparations was
typically 1.8 mg/mL as protein.
LDL-GAG Interaction Experiments
Experiments on the interaction between human plasma
LDL and arterial GAGs were performed essentially as de-
Cardoso and Mourao
Arterial Glycosaminogfycans and LDL
117
TABLE 1 . Total Hexuronic A d d and Hydroxyproline Contents in DIfterent Arteries
Artery
Abdominal Aorta
Thoracic Aorta
Iliac Artery
Pulmonary Artery
8.1 ±0.71
8.2±0.71
7.7±1.36
7.2±1.32
6.1-9.2
5.8-10.6
5.1-12.7
4.3-10.4
4
4
5
4
Mean±SEM
50.7±3.82
54.7±4.91
76.0± 11.51
69.5±6.84
Range
41.1-57.8
46.1-73.0
50.9-103.7
49.9-81.1
5
4
4
Hexuronic acid
Mean±SEM
Range
n
Hydroxyproline
n
5
Contents are given in micrograms per milligram of dry, defatted tissue. Hexuronic acid was measured on total
glycosaminoglycan extracts from tissue samples by a carbazole method.30 For the hydroxyproline assay, tissue
samples were hydrolyzed In 6 mol/L HCI and evaporated to dryness, and the residues, redissolved in water, were
assayed according to a chloramine T/p-dimethylaminobenzaldehyde method.31 n indicates number of individuals.
Downloaded from http://atvb.ahajournals.org/ by guest on June 14, 2017
scribed elsewhere23-33 and comprised the in vitro formation of
insoluble LDL-GAG complexes and LDL-affinity chromatography. Approximately 40 fig LDL (as protein) in 5 mmol/L
sodium acetate buffer, pH 6.0, containing 0.05 mol/L CaCl2
was mixed with variable quantities of GAGs in a final volume
of 1 mL, and after 10 minutes at 37°C, the absorbance was read
at 620 nm. About 90% of the LDL was insolubilized by the
maximal GAG concentration, and this was not affected by pH
within a range of 5.0 to 8.0. This proportion was determined by
a commercial cholesterol assay kit based on Allain et al36 and
was performed on the reaction mixture before addition of
GAG and on the LDL-GAG complex. For preparation of the
LDL-affinity columns, LDL was first coupled to cyanogen
bromide-activated Sepharose 4B following the standard procedure supplied with the product. Integrity of the immobilized
LDL in mounted columns (6x20 mm) was tested by its ability
to retain anti-apolipoprotein B antibody.22 Elution was accomplished by washing the column with 2 mL 0.1 mol/L EDTA and
then exhaustively with distilled water, after which 200-/ig
aliquots of GAG made in 0.05 mol/L CaCl2 were applied to the
column and eluted stepwise with 1 mL of NaG solution (0 to
0.25 mol/L) containing 0.05 mol/L CaCl2. GAGs in the eluates
were assayed for hexuronic acid30 and recovered by precipitation in three volumes of absolute ethanol at -20°C for 48
hours.
Quantitative experiments were performed with three replicates from which a mean value was obtained. Experiments
yielding densitometric profiles were conducted in three runs
for each sample. The depicted profiles are typical representatives from the replicates, whose variation has been found not
to exceed 5% around this typical profile in terms of peak
height and position. Quantitation of materials represented by
the peaks were made by estimating peak areas from these
typical profiles.
Differences among arteries for total hexuronic acid and
hydroxyproline contents were assessed by the Kruskal-Wallis
test, in which values of P<.05 were considered statistically
significant.
Results
Comparative Analysis of GAGs From Different
Human Arteries
The four arteries under study were not significantly
different in their total GAG and collagen contents as
measured by hexuronic acid and hydroxyproline, respectively (Table 1). A purified extract of total arterial GAGs
was obtained by papain digestion and cetylpyridinium
chloride precipitation, and ion-exchange chromatography
on DEAE-cellulose resolved this extract into three peaks,
identified as hyaluronic acid, heparan sulfate, and
dermatan+chondroitin 4/6-sulfate (Fig 1). These data,
combined with those from the degradation of galactosaminoglycans (dermatan sulfate+chondroitin 4/6-sulfate)
with specific GAG lyases (Table 2), permitted estimation
of the concentration of several GAG species (Table 3). It
can be seen that whereas there are no important differences in the contents of dermatan sulfate, the aortas have
on average 31% more chondroitin 4/6-sulfate than other
arteries when absolute values are considered. Furthermore, the pulmonary artery has approximately twice as
much hyaluronic acid as the other arteries, and the iliac
artery has a somewhat higher concentration of heparan
sulfate. These two arteries also gave different elution
profiles on DEAE-cellulose chromatography compared
with the aortas (Fig 1A through ID), indicative of possible
structural dissimilarities in the GAG chains.
The proportions of disaccharide units making up the
galactosaminoglycans obtained from DEAE-cellulose
chromatography were determined by chondroitin AC
and ABC lyase degradation, and the results are shown
in Table 2. Although the figures do not vary much, if
arteries are ordered according to decreasing 6-sulfated disaccharide (glucuronic acid and 6-sulfated
N-acetyl-D-galactosamine) contents, the series abdominal aorta=thoracic aorta>iliac artery=pulmonary artery is obtained. If, on the other hand, ordering is
based on the glucuronic acid-bearing, 4-sulfated disaccharide (glucuronic acid and 4-sulfated /V-acetyl-Dgalactosamine), approximately the same sequence is
obtained but in reverse order. The aortas have therefore the highest glucuronic acid-6-sulfated N-acetylD-galactosamine to glucuronic acid-4-sulfated N-acetyl-D-galactosamine ratio, whereas the pulmonary and
iliac arteries have the smallest. With respect to the
iduronic acid-bearing disaccharide, a similar pattern
is less evident.
A comparable disaccharide analysis was performed
with the heparan sulfate fraction recovered from the
DEAE column (Table 4). Here, though, the overall
proportions of disaccharides making up this GAG are
quite similar.
118
Arteriosclerosis and Thrombosis
Vol 14, No 1 January 1994
DS/CS
DS/CS
1.2
-®
A 0.59 M
4i \ 1
0.3
T "
0.8
0.2
*•*
0.8
HA
0.4
0
t
i
i
i
0.6
0.6
\
.•*••••»• •«• •
0.8
;
0.4
o.i
0.4 ^
JU 4 \
0.2
l_
i
1
0.2 I
0
!
E
m
1.2
Downloaded from http://atvb.ahajournals.org/ by guest on June 14, 2017
o.e
0.4
0
30
Fraction
60
90
0
30
60
90
Fraction number
number
HS DS CS
DS/CS-fraction
+ chondroitinase ABC
DS/CS-fraction
+ chondroitinase AC
DS/CS- fraction
+ buffer
HS-fraction
Total
FK3 1. Fractionation by DEAE-cellulose chromatography (A through D) and identification of the resulting fractions by agarose gel
electrophoresis (E) of the total glycosaminoglycans (QAGs) from the abdominal aorta (A), thoracic aorta (B and E), iliac artery (C), and
pulmonary artery (D). Recovered materials were identified as hyaluronic acid (HA), heparan sulfate (HS), and dermatan
sulfate+chondroitin 4/6-sulfate (DS/CS). Identifications were carried out as follows. HA is not detected on toluidine blue-stained agarose
gels and yields exclusively an unsaturated, nonsulfated disaccharide (Aglucuronic acid-W-acetyl-o-glucosamine) after chondroitin AC
lyase digestion (not shown). HS comigrates with the corresponding standard on agarose gel electrophoresis (E) and resists chondroitin
AC or ABC lyase digestion (not shown) but is degraded by heparitinases, forming unsaturated disaccharides shown in Table 4. The third
peak from the DEAE column contains both DS and C4/6-S as evidenced by a combination of agarose gel electrophoresis (E) with the
following procedures: incubation of this fraction wfth chondroitin AC lyase only degrades the GAG, which shows electrophoretlc
migration similar to that of standard C4/6-S, forms the unsaturated disaccharides described in Table 2, and leaves a peak that
comigrates with the standard of dermatan sulfate (E); finally, treatment with chondroitin ABC lyase degrades the GAGs with
electrophoretJc migration similar to that of C4/6-S and DS standards (E). All arteries gave similar anatytJc results. Profiles in E were
obtained by densitometry of toluidine blue-stained agarose gels; broken lines represent the electrophoretlc mobilities of standards of
HS, DS, and C4-S.
Cardoso and Mourao
TABLE 2.
Arterial Glycosaminoglycans and LDL
119
Proportions of Dlsaccharide Constituents of Arterial Galactosamlnoglycans
Artery
Dlsaccharide Units
Abdominal Aorta
Thoracic Aorta
lilac Artery
Pulmonary Artery
GteUA-GalNAc4S
13
17
20
18
GlcUA-GalNAc6S
50
50
45
43
ldUA-GalNAc4S
37
33
35
39
Relative proportions of disaccharide units were determined by degradation with chondroitin AC and ABC lyases
of the dermatan sulfate+chondroitin 4(4S)/6(6S)-sulfate peak from DEAE chromatography. The resulting degradation products were separated by paper chromatography on isobutyric acld/NH4OH, followed by silver staining and
quantitation by densitometry. See "Methods" for experimental details. GlcUA indicates glucuronic acid; GaJNac,
/V-acetyl-D-galactosamine; and IdUA, iduronic acid.
Downloaded from http://atvb.ahajournals.org/ by guest on June 14, 2017
Interaction Between GAGs From Various Arteries
and Plasma LDL
This was first analyzed by in vitro formation of
insoluble GAG-LDL complexes, whereby greater binding affinity was translated into greater turbidity of the
reaction mixture.22-33 GAG fractions from DEAE-cellulose chromatography of several arteries were added to
lipoprotein preparations purified from human plasma,
and the results are shown in Fig 2. Of the three GAG
fractions, only dermatan sulfate+chondroitin 4/6-sulfate interacted appreciably with LDL, although a clear
distinction between the different arteries could not be
made. Thus, affinity chromatography, a method enabling greater resolution in the analysis of GAG-LDL
interaction, was subsequently employed.
A column for affinity chromatography was prepared
by coupling human LDL to Sepharose 4B, and to this
column the dermatan sulfate+chondroitin 4/6-sulfate
fractions were applied (Fig 3). The proportions of
GAGs contained in each fraction into which the eluates
were divided are shown in Table 5. The elution profiles
have irregular contours and span a relatively wide range
of ionic strength, probably reflecting the heterogeneous
composition of the dermatan sulfate+chondroition 4/6sulfate fraction. But most importantly this figure reveals
marked differences among the elution profiles of the
four arteries, with the abdominal aorta having the
greatest binding affinity for LDL, since it contains the
highest proportion (25%) of GAGs in the fraction that
eluted at the highest ionic strength, together with lesser
amounts of material in the fractions of lower ionic
strength. This is followed in order of decreasing binding
affinity by the thoracic aorta, the iliac artery, and the
pulmonary artery. The latter two arteries have very
TABLE 3.
similar elution profiles, and their GAG contents eluted
in their entirety in fractions 1 and 2 (Fig 3, horizontal
brackets), which indicates that as a whole, these GAGs
have weaker affinities for LDL compared with those
from the aorta. A very pronounced difference in binding
affinity, for example, can be observed at 0.2 mol/L
NaCl. At this ionic strength, most or all of the applied
GAGs from the iliac and pulmonary arteries have
already eluted, whereas elution of GAGs from the
abdominal aorta is at its peak. The proportions of
GAGs contained in the fractions from the eluates
(Table 5) also show this difference and highlight the
absence of fraction 3 from the iliac and pulmonary
arteries.
The three fractions in the eluates from LDL-affinity
chromatography (Fig 3, horizontal brackets) were analyzed for molecular weight and relative contents of
dermatan sulfate and chondroitin 4/6-sulfate. The first
analysis is shown in Fig 4, which depicts PAGE slabs (Fig
4A) and their densitometric profiles (Fig 4B through
4D). For each fraction, the molecular weight of its
constituent GAGs do not vary much among arteries.
However, a progressive increase in molecular weight
toward fraction 3 is clearly noticeable, which means that
the dermatan sulfate+chondroitin 4/6-sulfate fraction
from all arteries is polydisperse and that in these dermatan sulfate+chondroitin 4/6-sulfate fractions, longer
GAG chains have greater binding affinities for LDL.
Last, the relative contents of dermatan sulfate and
chondroitin 4/6-sulfate in the fractions from LDLaffinity chromatography were determined. The results
are shown in Table 6, from which the following observations can be made: (1) chondroitin 4/6-sulfate is the
predominant GAG in all fractions from all arteries; (2)
dermatan sulfate is constituted mostly of polysaccharide
Relative and Absolute Contents of Various Glycosaminoglycans In Different Arteries
Hyaluronlc
Acid
Heparan
Sulfate
Dermatan
Sulfate
Chondroitin
4/6-Sutfate
Abdominal aorta
6.1 (0.5)
31.8(2.6)
23.2 (1.9)
38.9 (3.2)
Thoracic aorta
4.8 (0.4)
30.5 (2.5)
21.3(1.7)
43.2 (3.5)
Iliac artery
4.0 (0.3)
46.4 (3.6)
17.3(1.3)
32.3 (2.5)
Pulmonary artery
9.6 (0.7)
29.7 (2.2)
23.9(1.7)
36.8 (2.6)
Artery
Relative values are percents of total, whereas absolute values (figures in parentheses) are in micrograms of
hexuronic acid per milligram dry tissue. Proportions of hyaluronic acid, heparan sulfate, and dermatan
sulfate+chondroitin 4/6-sulfate were obtained by ion-exchange chromatography on DEAE-cellulose (Fig 1A through
1D). Proportions of dermatan sulfate and chondroitin 4/6-sulfate were obtained by analysis of unsaturated
disaccharides formed by chondroitin AC and ABC lyases. Absolute values were calculated from the mean total
hexuronic add concentrations (Table 1).
120
Arteriosclerosis and Thrombosis
TABLE 4.
Vol 14, No 1 January 1994
Proportions of DIsaccharide Constituents of Arterial Heparan Sulfates
Artery
Abdominal Aorta
Thoracic Aorta
lilac Artery
Pulmonary Artery
AUA-GlcNS, 6S
17
15
18
20
AUA-GlcNS
26
25
26
27
AUA-GlcNAc6S
17
16
20
13
AUA-GlcNAc
40
45
36
40
Heparltlna8e Products
Same methods were used as in Table 2, but with the heparan sulfate peak and heparitinases from Flavobacterium
heparlnum. A indicates unsaturated; UA, uronic acid; QlcNS, /V-sulfated glucosamine; 6S, 6-sutfated; and GlcNAc,
W-acetyt-D-glucosamine.
Downloaded from http://atvb.ahajournals.org/ by guest on June 14, 2017
chains with a comparatively high affinity for LDL, as the
major portion of this GAG eluted in fractions 2 and 3,
ie, the fractions of higher ionic strengths; (3) the relative
contents of chondroitin 4/6-sulfate, on the other hand,
decrease in inverse relation to the ionic strength, which
indicates that chains with a comparatively low affinity
for LDL are the major constituent of this GAG; (4) the
two aortas are nearly identical; besides the presence of
material still eluting in fraction 3, the aortas differ from
the other two arteries, in that their dennatan sulfate has
a higher binding affinity for LDL because the greatest
proportion of this GAG eluted in fraction 3 and only a
minor proportion eluted in fraction 1; (5) the iliac and
pulmonary arteries differ, in that the former has a
greater proportion of dermatan sulfate in fraction 2
than the latter. Fractions 1 through 3 from the thoracic
aorta were further analyzed by chondroitin AC and
ABC ryase degradation, which did not reveal detectable
amounts of nonsulfated or disulfated disaccharides (not
shown).
used in the present study were selected on the basis of
their known susceptibilities to develop such lesions,3 so
that vessels at high to low risk of lesion development
would be represented. Additionally, to minimize the
effects of aging, which alters arterial extracellular matrix37-38 and increases the severity39 and modifies the
distribution3 of atherosclerotic lesions, only arteries
from young adults were used. Thus, for the age group
from which the material was obtained, the abdominal
aorta is the artery where lesions occur most frequently, and it is followed not too distantly by the
thoracic aorta.
Fraction 1
.15
Discussion
To assess the involvement of GAGs in the regional
distribution of atherosclerotic lesions, the four arteries
,
•••
> m
•mill
.90
.15
0.4 -
•mill.
a •*>
< .15
0.3 -
0.2 0
50
0.1
.15
1.25
3.75
2.50
Glycosaminoglycon
—
5.0O
jug / m l
Fra 2. Line plot of in vitro formation of insoluble complexes of
arterial glycosamlnogtycan fractions and plasma low-density
lipoprotein. Dermatan sutfate+chondrortin 4/6-sutfate fractions
from the thoracic (•) and abdominal (o) aortas and from the iliac
(D) and pulmonary (•) arteries; heparan sulfate (A) and hyaluronlc add (A) fractions from the abdominal aorta. Heparan
sutfate and hyaluronic acid curves were similar for other arteries.
.nllll
N a C I - mM
FIG 3. Bar graph of low-density lipoprotein-affinity chromatography of dermatan sulfate+chondroitin 4/6-sulfate fractions from
the abdominal (A) and thoracic (B) aortas and from the iliac (C)
and pulmonary (D) arteries. The column was prepared and
eluted as described in "Methods." Eluates were pooled as
indicated by the horizontal bars, and the three fractions obtained
were then subjected separately to further analyses.
Cardoso and Mourao Arterial Glycosaminoglycans and LDL
121
TABLE 5. Relative Contents of Glycosaminoglycans In Fractions of Eluates From
LDL-Afflnlty Chromatography
Artery
Fraction
Abdominal Aorta
Thoracic Aorta
lilac Artery
Pulmonary Artery
1 (10-50)
38.6
41.4
44.2
42.1
2 (75-150)
36.1
44.7
52.7
57.9
3 (200-250)
25.4
13.9
3.0
The dermatan sulfate+chondroitin 4/6-sulfate fractions from ion-exchange chromatography were applied to
low-density lipoprotein (LDL)-affinity columns and then subjected to stepwise elution with solutions of increasing
sodium chloride concentration. Obtained elution fractions were pooled as fractions 1, 2, and 3 (see Fig 3), which
represent ranges of sodium chloride concentration In millimoles per liter (figures in parentheses). Values for arteries
are percentage of total eluted glycosaminoglycan.
The common iliac arteries are close to the thoracic
aorta, whereas the internal and external iliac arteries
are similar to the pulmonary artery, in that they are
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-4
I
*
A
-
E
o
.I,
5
o
Fraction:
Artery:
Fraction 1
10
9
4
-4
M.W. x 10
FIG 4. Pofyacryiamide gel electrophoresis (A) of the glycosaminoglycan fractions eluted from low-density lipoprotein-affintty
chromatography. Samples were run on 6%, 8x4-cm gels,
stained with toluidine blue, and scanned on a densitometer. The
resulting densftometric profiles for each of three fractions were
combined and are shown in B, C, and D. The symbol —
indicates abdominal aorta; — , thoracic aorta; • • •, iliac artery;
and
, pulmonary artery. M.W. Indicates molecular weight.
among the least susceptible. In terms of risk for atherosclerosis, the iliac artery samples in the present study
should therefore be situated between the thoracic aorta
and the pulmonary artery.
While the concentrations of total collagen and GAG
were approximately the same in the material studied
(Table 1), analysis of the contents of individual GAGs
revealed some differences (Table 3). The higher concentration of hyaluronic acid in the pulmonary artery
confirms previously published results.40 The aortas have
similar contents of dermatan sulfate but higher contents
of chondroitin 4/6-sulfate, and these GAGs have been
reported to have significant interactions with LDL.24-33
The values for dermatan sulfate are at variance with
those of Murata and Yokoyama,40 who detected lesser
amounts of this GAG in the pulmonary artery compared with the aorta. The iliac and pulmonary arteries
may have sulfated GAGs with different charge densities
from those of the aortas, as demonstrated by variations
in the elution profiles from ion-exchange chromatography (Fig 1A through ID). Still, with the exception of the
analysis of unsarurated disaccharides in the dermatan
sulfate+chondroitin 4/6-sulfate and heparan sulfate
chains (Tables 2 and 4), which does not imply variations
in degrees of sulfation, this has not been investigated
further.
Steric factors play a role in the interaction between
GAGs and LDL; as has been demonstrated, of the
isomeric forms of chondroitin 4/6-sulfate, it is the 6-sulfated form that binds strongly to LDL, whereas the
4-sulfated isomer shows little or no interaction.24 In this
respect, it is worth mentioning that both the incidence
and severity of atherosclerosis3-39 and the aortic concentration of chondroitin 6-sulfate37 increase with age. Thus,
these isomers may also influence the location of arterial
lesions, since the ratio of chondroitin 6-sulfate to chondroitin 4-sulfate decreases in an order corresponding to
decreasing risk for atherosclerosis (Table 2).
More elucidating results, however, were obtained in
the GAG-LDL interaction experiments performed with
the GAG fractions from ion-exchange chromatography.
Formation of insoluble GAG-LDL complexes (Fig 2), a
less discriminating technique, only confirmed the known
fact that arterial heparan sulfate and hyaluronic acid do
not bind LDL,33 without disclosing significant differences among the chondroitin 4/6-sulfate+dermatan sulfate fractions from the four arteries. LDL-affinity chromatography with these latter GAG fractions, on the
other hand, revealed that the four arteries differed
markedly (Fig 3 and Table 5), and their ordering
122
Arteriosclerosis and Thrombosis
Vol 14, No 1 January 1994
TABLE 6. Relative Contents of Dermatan Sulfate and Chondroitin 4/6-Sulfate In Fractions From
LOL-Afflnity Chromatography
Fraction 1
(10-50)
Artery
DS
Fraction 3
(200-250)
Fraction 2
(75-150)
CS
DS
CS
DS
CS
Abdominal aorta
5.2
94.8
21.6
78.4
37.0
63.0
Thoracic aorta
5.8
94.2
20.6
79.4
36.1
63.9
Iliac artery
12.9
87.1
46.1
53.9
Pulmonary artery
11.4
88.6
30.3
69.7
Proportions of dermatan sulfate (DS) and chondroitin sutfate (CS) for each fraction were determined by agarose gel
etectrophoresis followed by densitometry of totukJine blue-stained slabs. Fractions 1 through 3 represent ranges of salt
concentration in miilimoles per liter (figures in parentheses) and were obtained as described in Table 5.
Downloaded from http://atvb.ahajournals.org/ by guest on June 14, 2017
according to decreasing binding affinity matched the
ordering according to decreasing susceptibility to atherosclerosis. Other important differences were detected
in the subsequent analyses carried out on three fractions
obtained from affinity chromatography (Fig 3, horizontal brackets). PAGE revealed a positive correlation
between affinity for LDL and increasing molecular
weight, which was observed in all of four arteries (Fig
4). These data confirm as well as extend those of a
previous study that has demonstrated that chondroitin
4/6-sulfate-I-dermatan sulfate fractions of higher molecular weight from human thoracic aortas bind more
efficiently to LDL.23 However, the present results show
that for a given affinity-chromatogTaphy fraction, variations in molecular weight are minimal, so the effects of
this parameter as a determinant of atherosclerosis risk
should be more limited. For example, the abdominal
aorta is at higher risk than the thoracic aorta, and this is
reflected in the elution profiles from LDL-affinity chromatography. Yet analysis of molecular weight in the
eluates did not show appreciable differences between
these two arteries (Fig 4). Other aspects of GAG
composition that affect interaction with LDL may thus
be involved, and the degree of sulfation is an important
one.41 Alternatively or in addition, the greater propensity to atherosclerosis of the abdominal aorta in relation
to the thoracic aorta may be due to the fact that, so far
as GAGs are concerned, the former has nearly twice as
much eluted material in fraction 3 (Table 5). This
fraction has been shown herein to be composed of GAG
chains of higher molecular weight (Fig 4) and to be
enriched in dermatan sulfate (Table 6) compared with
the other two fractions. Earlier work has shown that
higher-molecular-weight GAGs 23 as well as dermatan
sutfate24 bind more avidly to LDL than lower-molecular-weight GAGs and either chondroitin 6- or 4-sulfate,
respectively. These two aspects of the interaction between GAGs and LDL thus explain the greater efficiency of GAG-LDL binding in fraction 3. Since the two
aortas have very similar contents of chondroitin 4/6sulfate and dermatan sulfate (Table 3), the abdominal
aorta has nearly two times more GAGs of higher affinity
to LDL than the thoracic aorta. In contrast, the pulmonary and iliac arteries either totally lack or have but a
small amount of fraction 3.
Therefore, the abdominal aorta and to a lesser extent
the thoracic aorta possess a particular GAG composition that potentially confers greater LDL binding capac-
ity to these arteries compared with the iliac and pulmonary arteries.
Reasons for these localized differences in biochemical composition and metabolism along the arterial bed
are still unclear. In a study with an atherosclerosissusceptible pigeon breed, Park et al42 have demonstrated that normal arteries from young animals already
have increased amounts of extracellular matrix proteins. That study also showed that the biosynthetic
activity for extracellular matrix components was normal
in other tissues, indicating that in the atherosclerosissusceptible breed, cells with altered phenotypes are
specifically located in arterial tissue. A similar conclusion was reached in investigations on GAGs from this
same breed, where alterations of synthesis/molecular
structure were detected.43'44 In different experiments,
an in vitro-elicited change of rabbit aortic smooth
muscle cells from a contractile to a less-contractile
phenotype was accompanied by an increase in the
synthesis of sulfated GAGs,45 and subsets of arterial
smooth muscle cells expressing different cytoskeletal
phenotypes were indeed demonstrated in atherosclerotic plaques and adjacent areas.46'47 Of the complex
array of mediators taking part in atherogenesis, growth
factors have a salient role,48 being present in lesions
since their earliest phases.49 Moreover, growth factors
are capable not only of inducing phenotypic changes in
arterial smooth muscle cells50 but also of increasing the
synthesis of chondroitin sulfate proteoglycan by these
cells.51 This proteoglycan also has longer GAG chains
and a higher content of the 6-sulfated isomer. In all of
these cases, modifications in cells and/or extracellular
matrixes might favor the development of atherosclerosis, and it is noteworthy that growth factor-induced
alterations in chondroitin sulfate proteoglycan5' correlate with our results for arteries at higher risk for
atherosclerosis. It may be speculated, therefore, that
lesion-prone areas in arteries are populated with cells of
analogous, potentially atherogenic phenotypes, whose
synthetic activities are or may become distinct from
those of cells in less susceptible areas.
In conclusion, the results of the present work demonstrated that GAGs from different human arteries
vary both in composition and in binding affinity to
plasma LDL. The consistency of these findings with
known data on the differential incidence of atherosclerotic lesions among arteries suggests that this heterogeneity of GAG composition participates in the multifac-
Cardoso and Mourao
tonal mechanisms that determine the susceptibility of a
given artery to atherosclerosis.
21.
Acknowledgments
This work was supported by Conselho Nacional de Desenvolvimcnto Cientifico e Tecnologico (CNPq: FNDCT and PADCT)
and Financiadora de Estudos e Projetos (FTNEP). The authors
thank Dr Helena B. Nader for the heparitinase degradations.
This work has been submitted to the Instituto de Biofisica Carlos
Chagas Filho, UFRJ, by Luiz E.M. Cardoso in partial fulfillment
of requirements for the degree of Doctor of Sciences.
References
Downloaded from http://atvb.ahajournals.org/ by guest on June 14, 2017
1. Roberts WC. Atherosclerotic risk factors —are there ten, or is
there only one? Atherosclerosis. 1992;97:S5-S9.
2. Duff GL, McMillan GC. Pathology of atherosclerosis. Am J Med.
1951;ll:92-108.
3. Roberts JC, Moses C, WiUtins RH. Autopsy studies in atherosclerosis: I. distribution and severity of atherosclerosis in patients
dying without morphologic evidence of atherosclerotic catastrophe. Circulation. 1959-.20-.511-519.
4. Schwartz CJ, Mitchell JRA. Observations on localization of
arterial plaques. Ci/r Res. 1962;ll:63-73.
5. Glagov S, Ozoa AK. Significance of the relatively low incidence of
atherosclerosis in the pulmonary, renal and mesenteric arteries.
Ann N YAcad Sri. 1968;149:940-955.
6. Fox JA, Hugh AE. Localization of atheroma: a theory based on
boundary layer separation. Br Heart J. 1966^28:388-399.
7. Zarvns CK, Giddens DP, Bharadvaj BK, Sottiurai VS, Mabon RF,
Glagov S. Carotid bifurcation atherosclerosis: quantitative correlation of plaque localization with flow velocity profiles and wall
shear stress. Ore Res. 1983;53:502-514.
8. Gibson CM, Diaz L, Kandarpa K, Sacks FM, Pasternak RC,
Sandor T, Feldman C, Stone PH. Relation of vessel wall shear
stress to atherosclerosis progression in human coronary arteries.
Arteriosder Thromb. 1993;13:310-315.
9. DePaola N, Gimbrone MA Jr, Davies PF, Dewey CF Jr. Vascular
endothelium responds to fluid shear stress gradients. Arteriosder
Thromb. 1992;12:1254-1257.
10. Cornhill JF, Barrett WA, Herderick EE, Mahley RW, Fry DL.
Topographic study of sudanophilic lesions in cholesterol-fed
minipigs by image analysis. Arteriosclerosis. 1985;5:415-426.
11. Wagner JD, St Clair RW, Schwenke DC, Shivery CA, Adams MR,
Clarkson TB. Regional differences in arterial low density lipoprotein metabolism in surgically postmenopausal cynomolgus
monkeys: effects of estrogen and progesterone replacement
therapy. Arteriosder Thromb. 1992;12:717-726.
12. Nielsen LB, Nordestgaard BG, Stender S, Kjeldsen K. Aortic
permeability to LDL as a predictor of aortic cholesterol accumulation in cholesterol-fed rabbits. Arteriosder Thromb. 1992;12:
1402-1409.
13. Gerrity RG, Richardson M, Somer JB, Bell FP, Schwartz CJ.
Endothelial cell morphology in areas of in vivo Evans blue uptake
in the aorta of young pigs: II. ultrastructure of the intima in areas
of differing permeability to proteins. Am J PathoL 1977;89:313 -334.
14. Kamanna VS, Vora S, Roh D, Kirschenbaum MA. Comparative
studies on acid cholesterol esterase in renal blood vessels and aorta
of control and hypercholesterolemic rabbits. Atherosclerosis. 1992;
94:27-33.
15. Svendsen E, Dregelid E, Eide GE. Internal elastic membrane in
the internal mammary and left anterior descending coronary
arteries and its relationship to intimal thickening. Atherosclerosis.
1990;83:239-248.
16. Srinivasan SR, Dolan P, Radhakrishnamurthy B, Pargaonkar PS,
Berenson GS. Lipoprotein-acid mucoporysaccharide complexes of
human atherosclerotic lesions. Biochim Biophys Acta. 1975;388:
58-70.
17. Berenson GS, Radhakrishnamurthy B, Srinivasan SR, Vijayagopal
P, Dalferes ER. Proteogrycans and potential mechanisms related
to atherosclerosis. Ann N YAcad ScL 1985;454:69-78.
18. Wight TN. Proteogh/cans in pathological conditions: atherosclerosis. Fed Prvc. 1985;44J81-385.
19. Hurt E, Bondjers G, Camejo G. Interaction of LDL with human
arterial proteogrycans stimulates its uptake by human monocytederived macrophages. J Upid Res. 1990-31:443-454.
20. Camejo G, Hun E, Wiklund O, Rosengren B, L6pez F, Bondjers
G. Modifications of low-density lipoprotein induced by arterial
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
Arterial Glycosaminoglycans and LDL
123
proteogrycans and chondroitin-6-sulfate. Biochim Biophys Ada.
1991;1096:253-261.
Camejo G, Hurt E, Romano M. Properties of lipoprotein complexes isolated by affinity chromatography from human aorta.
Bwmed Biochim Acta. 1985;44:389-401.
Mourao PAS, Pillai S, DiFerrante N. The binding of chondroitin
6-sulfate to plasma low density lipoprotein. Biochim Biophys Acta.
1981;674:178-187.
Alves CS, Mourao PAS. Interaction of high molecular weight
chondroitin sulfate from human aorta with plasma low density
lipoprotein. Atherosclerosis. 1988;73:113-124.
Christner JE, Baker JR. A competitive assay of lipoprotein:
proteoglycan interaction using a 96-well microtitration plate. Anal
Biochem. 1990;184:388-394.
Manley G, Hawksworth J. Distribution of mucoporysaccharides in
the human vascular tree. Nature. 1965^206:1152-1153.
Fischer GM, Llaurado JG. Collagen and elastin content in canine
arteries selected from functionally different vascular beds. Cur Res.
1966;19J94-399.
Scott JE. Proteogrycan-fibrillar collagen interactions. Biochem J.
1988;252:313-323.
Contri MB, Fornieri C, Ronchetti IP. Elastin-proteogrycan association revealed by cytochemical methods. Connect Tissue Res.
1985;13:237-249.
Cardoso LEM, Erlich RB, Rudge MC, Pera?oli JC, Mourao PAS.
A comparative analysis of the glycosaminoglycans from human
umbilical arteries in normal subjects and in pathological conditions
affecting pregnancy. Lab Invest. 1992;67:588-595.
Dische Z. A new specific color reaction of hexuronic acids. J Biol
Chan. 1947;167:189-198.
Bergman E, Loxley R. Two improved and simplified methods for
the spectrophotometric determination of hydroxyproline. Anal
Biochem. 1963^5:1961-1965.
Hilbom JC, Anastassiadis PA. Acrylamide gel electrophoresis of
acidic mucoporysaccharides. Anal Biochem. 1969;31:51-58.
Mourao PAS, Bracamonte CA. The binding of human aortic glycosaminoglycans and proteogh/cans to plasma low density lipoproteins. Atherosclerosis. 1984;50:133-146.
Nader HB, Porcionatto MA, Tersariol ILS, Pinhal MAS, Oliveira
FW, Moraes CT, Dietrich CP. Purification and substrate specificity of heparitinase I and heparitinase II from Flavobacterium
heparinum: analyses of the heparin and heparan sulfate degradation products by ' 3 C NMR spectroscopy. J Biol Chem. 1990;
265:16807-16813.
Havel RJ, Eder HA, Bragdon JH. The distribution and chemical
composition of ultracentrifugally separated lipoproteins in human
serum. / Clin Invest 1955^4:1345-1353.
Allain CC, Poon LS, Chan CSG, Richmond W, Fu PC. Enzymatic
determination of total serum cholesterol. Clin Chem. 1974;20:
470-475.
Toledo OMS, Mourgo PAS. Sulfated glycosaminoglycans of
human aorta: chondroitin 6-sulfate increase with age. Biochem
Biophys Res Commun. 1979;89:50-55.
Feldman S, Glagov S. Transmedial collagen and elastin gradients
in human aortas: reversal with age. Atherosclerosis. 1971;13:
385-394.
Spagnoli LG, Orlandi A, Mauriello A, Santeusanio G, Angelis C,
Lucreziotti R, Ramacci MT. Aging and atherosclerosis in the
rabbit: I. distribution, prevalence and morphology of atherosclerotic lesions. Atherosclerosis. 1991;89:ll-24.
Murata K, Yokoyama Y. High hyaluronic acid and low dermatan
sulfate contents in human pulmonary arteries compared to in the
aorta. Blood Vessds. 1988;25:1-11.
Sambandam T, Baker JR, Christner JE, Ekborg SL. Specificity of
the low density lipoprotein-grycosaminogrycan interaction. Arteriosder Thromb. 1991;ll:561-568.
Park H-S, Kniep AC, Smith SC, Robie SM, Smith EC, Yu SY,
Mackenzie JW, Scott GE, Boyd CD. Changes in vascular extracellular matrix accumulation reflect phenotypic differences
between the arterial wall of pigeons resistant and susceptible to the
development of spontaneous atherosclerosis. Connect Tissue Res.
1990:25:67-76.
Wight TN. Differences in the synthesis and secretion of sulfated
glycosaminoglycans by aorta explant monolayers cultured from
atherosclerotic-susceptible and -resistant pigeons. Am J PathoL
1980;101:127-142.
Rowe HA, Wagner WD. Arterial dermatan sulfate proteoglycan
structure in pigeons susceptible to atherosclerosis. Arteriosclerosis.
1985^5:101-109.
124
Arteriosclerosis and Thrombosis
Vol 14, No 1 January 1994
45. Merrilees MJ, Campbell JH, Spanidis E, Campbell GR. Glycosaminogh/can synthesis by smooth muscle cells of differing phenotype and their response to endotheliaJ cell conditioned medium.
Atherosclerosis. 1990;81:245-254.
46. Babaev VR, Bobryshev YV, Stenina OV, TararaV EM, Gabbiani
G. Heterogeneity of smooth muscle cells in atheromatous plaque
of human aorta. Am J PathoL 1990;136:1031-1042.
47. Ghiriato L, Scatena M, Chiavegato A, Zanello AMC, Guidolin D,
Pauletto P, Sartore S. Localization and smooth muscle cell composition of atherosclerotic lesions in Watanabe heritable hyperlipidemk rabbits. Arterioscler Thromb. 1993;13:347-359.
48. Klagsbrun M, Edelman ER. Biological and biochemical properties
of fibroblast growth factors: implications for the pathogenesis of
atherosclerosis. Arteriosclerosis. 1989;9:269-278.
49. Ross R, Masuda J, Raines EW, Gown AM, Katsuda S, Sasahara
M, Maiden LT, Masuko H, Sato H. Localization of PDGF-B
protein in macrophages in all phases of atherogenesis. Science.
199O;248:10O9-1012.
50. Morisaki N, Koyama N, Kawano M, Mori S, Umemiya K,
Koshilcawa T, Saito Y, Yoshida S. Human macrophages modulate
the phenotype of cultured rabbit aortic smooth muscle cells
through secretion of platelet-derived growth factor. Eur J Clin
Invest. 1992;22:461-468.
51. Schdnherr E, Jarvelainen HT, SandeU LJ, Wight TN. EffecU of
platelet-derived growth factor and transforming growth factor-/31
on the synthesis of a large verskan-like chondroitin sutfate proteogrycan by arterial smooth muscle cells. / Biol Chem. 1991^266:
17640-17647.
Downloaded from http://atvb.ahajournals.org/ by guest on June 14, 2017
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Glycosaminoglycan fractions from human arteries presenting diverse susceptibilities to
atherosclerosis have different binding affinities to plasma LDL.
L E Cardoso and P A Mourão
Arterioscler Thromb Vasc Biol. 1994;14:115-124
doi: 10.1161/01.ATV.14.1.115
Arteriosclerosis, Thrombosis, and Vascular Biology is published by the American Heart Association, 7272
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