© 1996 American Heart Association, Inc.
Articles |
Increased Expression of Genes for Platelet-Derived Growth Factor in Circulating Mononuclear Cells of Hypercholesterolemic Patients
From the Department of Biochemistry, University of Nottingham Medical School, Queen's Medical Centre, and the Department of Clinical Chemistry, City Hospital, Nottingham (C.B.M.), UK.
Correspondence to Dr M.A. Billett, Department of Biochemistry, University of Nottingham Medical School, Queen's Medical Centre, Nottingham NG7 2UH, UK.
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Abstract |
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Abstract Platelet-derived growth factor (PDGF) is implicated in the accumulation of smooth muscle cells in atherosclerotic lesions following monocyte migration through the vascular endothelium. We show here a 15- to 20-fold increase in expression of PDGF-A and -B genes (as measured by a quantitative reverse transcription–polymerase chain reaction assay of mRNA concentration) in circulating monocytes of hypercholesterolemic and hyperlipidemic patients compared with normocholesterolemic individuals. Strong positive correlations between PDGF-A and -B mRNA concentrations indicate that the two genes are coordinately regulated in mononuclear cells in both normal and hypercholesterolemic individuals. PDGF gene expression in patients correlates with concentrations of plasma total cholesterol and of low-density lipoprotein cholesterol, a proven risk factor for atherosclerosis. Activation of monocyte PDGF expression may be an important component of the atherosclerotic risk associated with raised cholesterol levels and may represent an essential step in the early stages of atherogenesis. However, the marked increases in PDGF mRNA levels in patients with modest hypercholesterolemia compared with normal subjects suggest that other factors are involved. The relationship of monocyte PDGF expression to other atherosclerotic risk factors and to the different stages of atherosclerosis needs to be carefully evaluated.
Key Words: atherosclerosis • platelet-derived growth factor-A and -B • mRNA • human blood mononuclear cells • hypercholesterolemia
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Introduction |
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TopAbstractIntroductionMethods Results Discussion References |
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PDGF is a 28- to 35-kD glycoprotein consisting of two polypeptide chains held together through disulfide bridges. The two chains, A and B, are encoded by separate genes found on human chromosomes 7 and 22, respectively.1 The dimeric protein is biologically active in each of three isomeric forms, AA, AB, and BB, suggesting independent regulation of the two genes.1 Responsive tissues may contain two forms of receptor,
and ß, each with different
affinities for A and B chains.1 PDGF is synthesized in
many cell types besides platelets and is a potent mitogen and
chemotactic agent for cells of mesenchymal origin, which include those
of the vasculature.1 2 3 It is thought to
play a fundamental
role in inflammation and atherogenesis, processes characterized by cell
proliferation.3 Thus, endothelial damage
causes the release of cytokines, which increase the adherence
and subsequent migration of monocytes to the
subendothelial space, where they start to
differentiate into macrophages.3 Besides
accumulating massive amounts of lipid from lipoproteins and eventually
forming foam cells, activated macrophages secrete PDGF,
as do smooth muscle cells, endothelial cells, and
platelets.1 4 Interestingly, nonactivated
monocytes only express the PDGF genes at a very low rate.5
PDGF in turn stimulates the migration and proliferation of medial
smooth muscle cells into the intima, where they too can accumulate
lipoprotein-borne lipid. Increased deposition of extracellular
matrix components accompanies this cell proliferation and foam cell
formation, gradually giving rise to the fibrous plaque characteristic
of atheroma.3 Although sophisticated
ultrasound and magnetic resonance imaging techniques are being
developed as noninvasive methods for measuring human atherosclerotic
lesions in accessible vessels such as the carotid
artery,6 7 there are no simple, routine, noninvasive
methods currently available for the detection of the early stages of
atheroma or its development in the whole body. A greater
understanding of the essential events occurring in the circulation that
reflect the initiation of atherogenesis would therefore be valuable in
this respect. Since activation of monocytes and the synthesis of PDGF
may represent such events, we have measured the expression of
PDGF A and B genes in circulating mononuclear cells in normal
individuals and hypercholesterolemic subjects at
risk for atherosclerosis. |
Methods |
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Subjects
Eight healthy normocholesterolemic subjects were chosen from staff within this institute. FH patients were classified by raised plasma and LDL cholesterol, the presence of tendon xanthomas, and having first-degree relatives with premature ischemic heart disease.8 Since no analyses of LDL receptor genotype were performed, this classification is provisional. Indeed, analysis of these patients for the Arg3500
Gln FDB mutation by a
modification of the Schuster et al9 method indicated that
patient FH4 carried an FDB mutation. Hyperlipidemic
patients were a heterogeneous group with
hypercholesterolemia and variable
hypertriglyceridemia who did not satisfy
the criteria for FH. Plasma total cholesterol and triacylglycerol concentrations were measured enzymatically using Olympus reagents (catalog No. 66805 and 66813). HDL cholesterol was determined after precipitation of apoB-containing lipoproteins with phosphotungstic acid and magnesium chloride.10 LDL cholesterol concentrations were calculated, where possible, using the Friedewald et al11 equation.
Mononuclear Cell RNA Isolation
Peripheral blood mononuclear
cells were isolated
using Histopaque-1077 (Sigma)12 and comprised on average
91% lymphocytes, 5% monocytes, and <3% neutrophils. In some
experiments, monocytes were removed from mononuclear preparations by
incubation in RPMI 1640 medium on plastic culture plates for 2 hours at
37°C; nonadherent cells consisted solely of lymphocytes. No attempt
was made to analyze monocytes directly, in view of the known
effect of adherence on monocyte PDGF expression.13 Total
cellular RNA was purified from mononuclear cells by the method of
Chomczinski and Sacchi.14
Quantitative RT-PCR
Quantitation of PDGF mRNA by RT-PCR was
essentially according to
Wang et al.15 Reverse transcription was performed with 1
µg total cellular RNA, 5x103 to
2x106 copies AW109 cRNA as internal standard
(Perkin-Elmer), 50 mmol/L Tris-HCl at pH 8.3, 75 mmol/L KCl, 3 mmol/L
MgCl2, 10 mmol/L DTT, 10 U RNasin (Promega), 5
µmol/L random hexanucleotide primers (Pharmacia),
0.125 mmol/L dNTPs, and 200 U Superscript Moloney murine leukemia virus
reverse transcriptase (GIBCO-BRL), in a total volume of 20 µL at
37°C for 2 to 5 hours. Serial 1:3 dilutions of the cDNA products
were amplified with 0.5 µmol/L PDGF-A or -B specific
primers,15 5 µC [-32P]dCTP, 0.025
mmol/L dNTPs, 10 mmol/L Tris-HCl at pH 9.0, 50 mmol/L KCl, 1.5 mmol/L
MgCl2, 0.1% Triton X-100, and 2.5 U Taq
DNA polymerase (Promega), in a volume of 100 µL for 25 cycles (0.5
minute at 94°C, 2 minutes at 55°C, and 3 minutes at 72°C). PCR
products were separated by electrophoresis on 3% Nusieve 3:1
agarose gels (FMC). Products of PDGF-A mRNA template (225 bp) and
AW109 cRNA (301 bp) or PDGF-B mRNA (217 bp) and AW109 cRNA (300 bp)
were cut from the gel, and 32P was quantitated by Cerenkov
counting. Linear double log plots of counts per minute against amounts
of template were drawn15 and numbers of copies of PDGF-A
or -B mRNA in total cellular RNA determined by equating with the number
of molecules of AW109 cRNA that generated the same amount of
radioactive PCR product (Fig 1
). Double log plots
for AW109 cRNA and each mRNA gave parallel straight-line plots,
indicating that the amplification efficiency was the same for both
templates. Corrections were made for differing percent
guanine+cytosine contents of each amplified template. Small
variations in amounts of RNA template were corrected by independently
measuring the poly(A) content of each sample by dot blot hybridization
against oligo(dT)18,16 and results were
expressed as copies mRNA per nanogram total cellular RNA, assuming 0.02
ng poly(A)/ng total cellular RNA.16 This quantitative
RT-PCR assay was found to be reproducible. Repeat amplifications of one
cDNA sample gave mRNA concentrations differing by ±3.5%. Reassay of
mononuclear PDGF-B mRNA from one individual on three occasions over 12
months gave values differing by ±9.5%.
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Statistical Analysis
Linear correlation coefficients were
calculated using CA Cricket
Graph III software (Computer Associates International Inc).
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Results |
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TopAbstractIntroductionMethods Results Discussion References |
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Concentrations of mRNA for PDGF-A and PDGF-B in PBMN were measured using the quantitative RT-PCR assay developed by Wang et al.15 This procedure depends on the inclusion in the RT-PCR reaction of known amounts of an internal RNA standard (pAW109 RNA) that can be amplified concurrently with the specific mRNA, using the same PCR primers but generating a product of a different size. The internal standard allows correction for variable amplification efficiency. In this system, PDGF-specific primers amplify regions of the cDNA from exon 3 to the exon 4/5 junction (PDGF-A) or between exon 3/4 and exon 4/5 (PDGF-B) and thus will detect all forms of mRNA transcript containing exon 4 of each PDGF gene, including the alternatively spliced forms of PDGF-A mRNA.17 18
PDGF mRNA levels in normal and hypercholesterolemic
individuals were compared. Hypercholesterolemic
patients were classified as either FH, on the basis of clinical data
and family history, or as HL, showing
hypercholesterolemia and variable
hypertriglyceridemia. Clinical details of
patients analyzed are given in Table 1, together
with information on normal subjects, and corresponding blood lipid
measurements are given in Table 2. In addition to
hypercholesterolemia, other positive risk
factors for coronary heart disease (eg, hypertension, smoking)
were associated with some individuals. Presence or absence of overt
vascular and other diseases and medication is also indicated in Table
1. Typical results of analyses of PDGF-A and -B mRNAs in one
normal and one hypercholesterolemic individual are
presented in Fig 1
. Low concentrations of PDGF-A and PDGF-B
mRNA were present in PBMN of eight
normocholesterolemic individuals (Table 2). In
contrast, hyperlipidemic patients showed an approximate
15-fold and FH patients an approximate 20-fold increase in amounts of
both mRNA species (Table 2). The FDB patient carrying a
mutation in the
region of apoB-100, which interacts with the LDL
receptor,9 had PDGF mRNA levels similar to other members
of the FH group. Preliminary data suggest that >90% of PDGF-A mRNA in
these patients is the short form, lacking exon
6.17 18
Since it is known that PDGF gene expression is rapidly induced when
monocytes adhere to plastic surfaces,13 no attempt was
made to isolate monocytes from PBMN preparations for direct
determination of their PDGF mRNA concentrations. However, when
monocytes from both normocholesterolemic and
hypercholesterolemic individuals were allowed to
adhere to plastic, the PDGF-A and PDGF-B mRNA content of the
nonadherent cells (lymphocytes) was undetectable, whereas mRNA
concentrations for LDL receptor15 and HMG CoA
reductase15 were similar to those of total PBMN (data not
shown). This confirms that the majority of both PDGF-A and PDGF-B mRNA
detected in PBMN of normocholesterolemic and
hypercholesterolemic individuals are derived from
monocytes. PDGF-A and -B mRNA species are not detectable in
granulocytes.5
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Although concentrations of PDGF-A and PDGF-B mRNA tended to be higher
in the FH group than the hyperlipidemics (Table 2),
there was in fact a better correlation with plasma
cholesterol levels than with this patient classification
per se. Indeed, clear positive correlations were seen for both mRNA
species with plasma total cholesterol (PDGF-A mRNA:
r=.890, P<.001; and PDGF-B mRNA:
r=.846, P<.001; Fig 2) and with
LDL cholesterol levels (PDGF-A mRNA: r=.888,
P<.001; and PDGF-B mRNA: r=.876,
P<.001; data not shown) when
normocholesterolemic and
hypercholesterolemic individuals were
analyzed together. When
hypercholesterolemic individuals were considered
alone, significant positive correlations of mRNA concentrations with
plasma total cholesterol (PDGF-A mRNA: r=.879,
P<.001; and PDGF-B mRNA: r=.665,
P<.05; data not shown) and with LDL cholesterol
(PDGF-A mRNA: r=.874, P<.001; and PDGF-B mRNA:
r=.728, P<.05; data not shown) were again
apparent. Corresponding positive correlations for
normocholesterolemic individuals failed to reach
significance, although PDGF-A mRNA did show a weak positive correlation
with the ratio of LDL cholesterol to HDL
cholesterol (r=.746, P<.05).
Stronger positive correlations were apparent between this ratio and
PDGF-A and -B mRNA levels for hypercholesterolemic
individuals alone (r=.772, P<.01 and
r=.746, P<.01, respectively). All of the above
correlations were also significant at the 1% or 5% level when FH
patients were analyzed separately. Interestingly, PDGF-A and -B
mRNA levels also demonstrated weak but significant negative
correlations with HDL cholesterol concentrations, when all
subjects were considered together (PDGF-A mRNA:
r=-.472, P<.05; and PDGF-B mRNA:
r=-.547, P<.05; data not shown).
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represents
normocholesterolemic individuals; 
, HL
individuals; and 
, FH individuals.
Although the data in Fig 2 demonstrate significant
correlations
of mononuclear cell PDGF mRNA levels with plasma total
cholesterol, closer examination of Fig 2A, in particular,
suggests the presence of a threshold effect. Thus, whereas among normal
individuals, there is a modest rise in PDGF-A mRNA as total
cholesterol increases from 3.8 to 5.2 mmol/L, the lowest
hypercholesterolemic patient
(cholesterol, 6.0 mmol/L) has at least a 10-fold higher
PDGF-A mRNA level. This implies that the greatly increased PDGF mRNA
concentrations in mononuclear cells of
hypercholesterolemic patients might at least in
part be associated with an additional confounding factor other than
cholesterol. This factor would be present in patients
but not normal subjects and might explain the apparent discontinuity in
the relationship between cholesterol and PDGF mRNA. We have
therefore investigated further potential correlations of PDGF mRNA
levels with other risk factors for coronary heart disease
(hypertension, smoking), or with age, gender, overt vascular or other
disease, plasma triglyceride concentration, and medication.
Among hypercholesterolemic subjects, a negative
correlation was apparent, which just reached significance at the 5%
level, between age and both PDGF mRNA concentrations. However, this was
heavily influenced by the very high mRNA levels in the young, severely
hypercholesterolemic patient FH7, and if data from
this individual were excluded, no significant correlation was
observed. No significant correlation with age was apparent for PDGF
mRNA levels in normal subjects. Similarly, PDGF mRNA levels showed no
correlation with gender in normal or
hypercholesterolemic subjects, although the small
number of female subjects in this study precludes definitive
conclusions. Plasma triglyceride concentration showed weak
positive correlations with PDGF-A and PDGF-B mRNA levels in normal
individuals (r=.773, P<.05; and
r=.776, P<.05, respectively), but no correlation
was evident among hypercholesterolemic subjects.
None of the other factors mentioned above showed significant
correlation with PDGF mRNA concentrations whether subjects were
considered as one group or separately as
normocholesterolemic and
hypercholesterolemic groups. In particular, the two
patients with evidence of cardiovascular disease (HL5
and FH7) and one who developed symptoms subsequent to sampling (FH6)
exhibited PDGF mRNA concentrations similar to other,
asymptomatic hypercholesterolemic
patients. Although definitive conclusions on the association of
smoking, hypertension, or vascular disease itself with PDGF mRNA levels
would require a study of larger subject groups for each category, the
present data suggest that plasma cholesterol is the
only individual variable showing significant correlation with PDGF
expression in the subjects examined here.
In both normocholesterolemic and
hypercholesterolemic subjects, there was a strong
positive correlation between the levels of PDGF-A and PDGF-B mRNA
species present in blood mononuclear cells (Fig 3).
This was apparent whether all subjects were considered together
(r=.901, P<.001) or
normocholesterolemic and
hypercholesterolemic subjects were analyzed
separately (r=.796, P<.01; and
r=.693, P<.01, respectively). This finding
indicates that the two genes are coordinately regulated in mononuclear
cells in both normal and hypercholesterolemic
individuals.
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All three isoforms of PDGF can upregulate LDL receptor mRNA, and
PDGF-BB can upregulate HMG CoA reductase mRNA in fibroblasts and smooth
muscle cells.19 It is, therefore, interesting to note that
in the mononuclear cells analyzed in the present
experiments, there were strong negative correlations between mRNA
levels for PDGF-A and -B on the one hand, and mRNA levels for LDL
receptor and HMG CoA reductase (also assayed by the Wang
procedure15 ) on the other, when data for all individuals
were considered together (Table 3). These reciprocal
relationships are consistent with our findings that mononuclear
cell mRNA levels for both HMG CoA reductase and LDL receptor negatively
correlate with plasma total and LDL cholesterol (see
Reference 20 and M.A.B. et al, 1995, unpublished observations), whereas
PDGF mRNA levels positively correlate with these parameters
(see above and Fig 2).
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Discussion |
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We have demonstrated 15- to 20-fold increases, relative to normal individuals, in the expression of PDGF-A and PDGF-B genes (reflected in mRNA levels) in circulating monocytes of HL and FH patients. These measurements of PDGF mRNA concentration were performed after isolation of mononuclear cells by density gradient centrifugation of blood, but with no further incubation. Although these are not strictly direct in vivo measurements, the very clear differences in PDGF mRNA observed between hypercholesterolemic and normocholesterolemic individuals argue strongly that the mononuclear cell isolation procedure has not distorted the data and that they reflect real differences in vivo.
Northern blotting techniques could not detect PDGF-A or PDGF-B mRNA in
freshly isolated, unstimulated monocytes,4 5 but
PDGF-B
mRNA was detectable after adherence of monocytes to a plastic
surface.13 Bacterial lipopolysaccharide,
interferon-
, and phorbol esters have been variously shown to
stimulate PDGF-A and/or PDGF-B mRNAs in monocyte-derived
macrophages and in the monocytic cell line
U937.1 15 21 22 23
More recently, sensitive RT-PCR assays
detected low levels of both PDGF-A and PDGF-B mRNA in circulating
monocytes, but not granulocytes, of normal
individuals.5
The increases in PDGF mRNA levels observed in the present work
could be due to an increase in transcription, a decrease in mRNA
degradation, or a combination of both. Many mitogens,
cytokines, and other factors, including transforming growth
factor-ß, tumor necrosis factor-, interleukin-1, and shear stress,
can stimulate PDGF gene
expression,1 22 24 25 whereas
others (eg, 
3 fatty acids) decrease
expression.1 5 22 26 Although
some of these factors
modulate PDGF mRNA stability,26 most agents directly
affect PDGF gene
transcription.1 22 24 25
Whereas the two
PDGF genes are independently regulated, some agents have similar
effects on both genes.1 22 We have clear evidence in
the
present work of coordinate regulation of PDGF-A and PDGF-B genes in
mononuclear cells of normal and
hypercholesterolemic patients.
Although we do not know the primary stimulus for PDGF expression in these patients, the simplest explanation would be a direct positive upregulation by cholesterol or a metabolite thereof. The data presented here are consistent with such a direct effect, particularly through LDL cholesterol, eg, positive correlations of PDGF-A and -B mRNA with total and LDL cholesterol (which are significant whether all subjects, hypercholesterolemic, or FH patients alone are considered) and negative correlations with LDL receptor and HMG CoA reductase mRNAs and with HDL cholesterol. Indeed, there is some preliminary evidence that cholesterol oxides (25-hydroxycholesterol, 7-ketocholesterol) can increase PDGF mRNA levels in cultured rabbit aortic smooth muscle cells.27 However, it is equally possible that some other factor is independently responsible for both the hypercholesterolemia and the raised PDGF mRNAs or that hypercholesterolemia initiates alterations in cytokine signaling between endothelium, monocytes, lymphocytes, or other cells, which cause induction of PDGF expression in monocytes. The potential for involvement of cytokines is illustrated by the recent demonstration that LDL cholesterol levels in children correlate with the number of cells in different T lymphocyte subsets.28
Furthermore, the correlations of cholesterol concentration
with PDGF-A mRNA show a sudden sharp increase in mRNA level between
normal and hypercholesterolemic individuals,
indicative of a threshold effect (Fig 2A). In the case of
PDGF-B mRNA,
however, the transition is more gradual. In view of the evidence for
the involvement of PDGF in atherogenesis,3 one might
presume that the increased PDGF mRNA levels in
hypercholesterolemic patients reflect the degree of
vascular damage in each individual and when added to the
cholesterol correlation might produce the apparent
threshold effect. However, many factors can influence the atherogenic
process, positively or negatively, including LDL and HDL
cholesterol, age, duration of
hypercholesterolemia, medication (especially
lipid-lowering drugs such as statins), smoking, hypertension, and
others.3 Only two patients in the current study had
established vascular disease, although one asymptomatic
patient developed symptoms a year after sampling. Nevertheless, it is
quite possible that active or early forms of atherogenesis were
present in other individuals yet were not extensive enough to cause
overt symptoms. Thus, the increased PDGF expression might reflect
vascular pathology and any other factors contributing to it. However,
in the present work, the only individually identifiable factor that
showed significant correlations with PDGF mRNA levels in
hypercholesterolemic subjects was
cholesterol itself.
Previous data on PDGF protein concentrations in plasma or serum from hypercholesterolemic patients are limited. One study measured PDGF in plasma indirectly and found a twofold increase in hypercholesterolemic patients with proven coronary atherosclerosis.29 In a similar study, plasma PDGF was found to correlate with the number and severity of distinct stenoses in atherosclerotic patients.30 A further study found a twofold increase in PDGF in serum of hypercholesterolemic patients, although there was no significant correlation between serum PDGF and total cholesterol in individuals.31 Whereas the majority of PDGF in serum is believed to reflect release from platelets,1 2 30 31 the much lower concentrations in plasma may be derived from platelets, monocytes/macrophages, or endothelial cells. There have been reports of increases in plasma PDGF levels in chronic arterial obstructive disease32 and in the coronary circulation, but not the aortic root, in patients with unstable angina.33 However, these observations are complicated by uncertainty as to the source of the PDGF and by suggestions that plasma levels are affected by changes in consumption of PDGF by damaged or repairing tissues.32 33
Little is known about the regulation of PDGF mRNA translation,
postranslational processing, or secretion of the mature PDGF
protein.1 However, the parallel effects of dietary fish
oil supplementation on concentrations of monocyte PDGF mRNAs and on
plasma PDGF protein levels suggest that monocyte mRNA levels may well
reflect PDGF protein production.5 34 The
half-life of PDGF in the circulation is no more than 2
minutes,1 2 indicating rapid uptake into target
tissues or
binding to other plasma proteins, such as
2-macroglobulin, or to proteoglycans on the
endothelium.35 Our preliminary
analysis of the different spliced variants of PDGF-A mRNA
suggests that monocytes of hypercholesterolemic
patients would predominantly produce the short form of PDGF-A protein,
lacking the exon-6 encoded retention motif, which mediates binding to
proteoglycans.35 However, since mature PDGF-B contains a
constitutive retention motif, it seems likely that PDGF-AB and -BB
will, at least, bind to proteoglycans of the extracellular matrix. In
this context, estimates of the rate of PDGF production from
mRNA concentrations in cells may well be more informative than
monitoring steady state levels of the protein in the circulation.
Furthermore, whether or not the elevated PDGF mRNA levels that we have
observed are reflected in increased PDGF protein in the circulation,
they serve as a striking marker for monocyte activation in these
patients.
Increased expression of both PDGF-A and PDGF-B genes is detected in
human atherosclerotic carotid
artery.3 4 15 Immunochemical
and in situ hybridization analyses indicate that smooth muscle
cells are the main site of PDGF-A mRNA, and macrophages, which
contain little or no lipid, are the major site of PDGF-B mRNA in
atherosclerotic lesions.3 4 Lipid-laden foam cells
appear to contain little PDGF-B, consistent with the
observation that oxidized LDL inhibits PDGF-B gene expression in
monocyte-derived macrophages.4 21 The
consensus view is therefore emerging that during development of
atherosclerotic lesions, differentiating macrophages release
PDGF-B, which acts as a chemoattractant to smooth muscle cells
migrating into the intima.3 Cytokines, such as
interleukin-1, tumor necrosis factor-, and transforming growth
factor-ß, released from activated macrophages induce
PDGF-B gene expression in endothelial cells and PDGF-A
gene expression in smooth muscle cells. Both PDGF-AA and PDGF-BB could
then stimulate smooth muscle cell proliferation in the
intima.3 Furthermore, in both humans and animal models,
PDGF is required for smooth muscle cell accumulation during
restenosis after arterial balloon
angioplasty.36 37 Thus, PDGF is seen to have a
pivotal
role in the fibroproliferative process of
atherosclerosis.3
It has been claimed, on the basis of experimentally induced atherosclerotic lesions in animals, that continuous recirculation of lipid-laden macrophages between lesions and bloodstream occurs.3 4 If this is so, our results might reflect recirculation of activated monocytes/macrophages prior to lipid loading, since this process is thought to downregulate PDGF expression. However, current evidence suggests that macrophages in atherosclerotic lesions contain predominantly PDGF-B mRNA, whereas our mononuclear cells contain similar concentrations of both PDGF-A and -B mRNAs. Whatever the cause of the increased PDGF expression in circulating mononuclear cells of hypercholesterolemic individuals, it seems likely that it represents some form of monocyte activation that may possibly be associated with early stages in the generation of lesions in the vessel wall/vasculature. It will clearly be important to determine whether elevated PDGF expression in circulating monocytes is associated with other positive risk factors for atherogenesis (eg, hypertension, smoking), especially in the absence of hypercholesterolemia, and to establish whether there is a clear relationship between monocyte PDGF expression and stages of atherosclerosis in larger groups of patients with differing severity of vascular disease. In the present studies, plasma cholesterol was the only individual variable correlating significantly with monocyte PDGF mRNA. It seems likely that elevated monocyte PDGF expression is a significant component of the mechanism that leads to increased atherosclerotic risk in hypercholesterolemia.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This work was supported in part by CORDA, the heart charity.
Received April 19, 1995; accepted November 20, 1995.
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References |
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TopAbstractIntroductionMethods Results Discussion References |
|---|
- Khachigian LM, Chesterman CN. Platelet-derived growth factor and its receptor: structure and roles in normal growth and pathology. Platelets. 1991;4:304-315.
- Ross R, Raines EW, Bowen-Pope DF. The biology of platelet derived growth factor. Cell. 1986;46:155-169. [Medline] [Order article via Infotrieve]
- Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature. 1993;362:801-809. [Medline] [Order article via Infotrieve]
-
Ross R, Masuda J, Raines EW, Gown AM, Katsuda S,
Sasahara M, Malden LT, Masuko H, Sato H. Localization of PDGF-B
protein in macrophages in all phases of atherogenesis.
Science. 1990;248:1009-1012.
[Abstract/Free Full Text] -
Kaminski WE, Jendraschak E, Kiefl R, von Schacky
C. Dietary 3 fatty acids lower levels of
platelet-derived growth factor mRNA in human mononuclear
cells. Blood. 1993;81:1871-1879.
[Abstract/Free Full Text] - Crouse JR, Thompson CJ. An evaluation of methods for imaging and quantifying coronary and carotid lumen stenosis and atherosclerosis. Circulation. 1993;87(suppl II):II-17-II-33.
- Skinner MP, Yuan C, Mitsumori L, Hayes CE, Raines EW, Nelson JA, Ross R. Serial magnetic resonance imaging of experimental atherosclerosis detects lesion fine structure, progression and complications in vivo. Nat Med. 1995;1:69-73. [Medline] [Order article via Infotrieve]
- Goldstein JL, Brown MS. Familial hypercholesterolemia. In: Scriver CR, Beaudet AL, Sly WS, Valle D, eds. Metabolic Basis of Inherited Disease. 6th ed. New York, NY: McGraw-Hill Publishing Co; 1989:1215-1250.
- Schuster H, Rauh G, Muller S, Keller C, Wolfram G, Zollner N. Allele-specific and asymmetric polymerase chain reaction amplification in combination: a one-step polymerase chain reaction protocol for rapid diagnosis of familial defective apolipoprotein B-100. Anal Biochem. 1992;204:22-25. [Medline] [Order article via Infotrieve]
- Burstein M, Scholnick HR, Morfin R. Rapid method for the isolation of lipoproteins from human serum by precipitation with polyurias. J Lipid Res. 1970;11:583-595. [Abstract]
- Friedewald WT, Levy RI, Fredrickson DS. Estimation of the concentration of low-density lipoprotein cholesterol in plasma, without use of the ultracentrifuge. Clin Chim Acta. 1972;71:397-402.
- Boyum A. Isolation of mononuclear cells and granulocytes from human blood: isolation of mononuclear cells by one centrifugation, and of granulocytes by combining centrifugation and sedimentation at 1g. Scand J Clin Lab Invest. 1968;21(suppl 97):77-89.
-
Shaw RJ, Doherty DE, Ritter AG, Benedict SH, Clark
RAF. Adherence-dependent increase in human monocyte PDGF(B)
mRNA is associated with increases in c-fos,
c-jun, and EGR2 mRNA. J Cell
Biol. 1990;111:2139-2148.
[Abstract/Free Full Text] - Chomczinski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987;162:156-159. [Medline] [Order article via Infotrieve]
-
Wang AM, Doyle MV, Mark DF. Quantitation of mRNA
by the polymerase chain reaction. Proc Natl Acad Sci
U S A. 1989;86:9717-9721.
[Abstract/Free Full Text] - Harley CB. Hybridization of oligo(dT) to RNA on nitrocellulose. Genet Anal Tech Appl. 1987;4:17-22.
-
Bonthron DT, Morton CC, Orkin SH, Collins T.
Platelet-derived growth factor A chain: gene structure,
chromosomal location and basis for alternative mRNA splicing.
Proc Natl Acad Sci U S A. 1988;85:1492-1495.
[Abstract/Free Full Text] - Sanchez A, Chesterman CN, Sleigh MJ. Novel human PDGF A gene transcripts derived by alternative mRNA splicing. Gene. 1988;98:295-298.
-
Roth M, Emmons LR, Perruchoud A, Block LH.
Expression of the low density lipoprotein receptor and
3-hydroxy-3-methylglutaryl coenzyme A reductase genes are stimulated by
recombinant platelet-derived growth factor isomers.
Proc Natl Acad Sci U S A. 1991;88:1888-1892.
[Abstract/Free Full Text] - Alrokayan SAH, Marenah CB, White DA, Billett MA. Quantitation of HMG-CoA reductase mRNA levels in peripheral blood mononuclear cells of normal individuals and patients with altered lipoprotein metabolism. Biochem Soc Trans. 1993;21:375S. [Medline] [Order article via Infotrieve]
-
Malden LT, Chait A, Raines EW, Ross R. The
influence of oxidatively modified low density lipoproteins on
expression of platelet-derived growth factor by human
monocyte-derived macrophages. J
Biol Chem. 1991;266:13901-13907.
[Abstract/Free Full Text] - Kaetzel DM, Coyne DW, Fenstermaker RA. Transcriptional control of the platelet-derived growth factor subunit genes. Biofactors. 1993;4:71-81.[Medline] [Order article via Infotrieve]
- Li SR, Forster L, Anggard E, Ferns G. The effects of LPS and probucol on interleukin-1 and platelet-derived growth factor gene expression in the human monocytic cell line U-937. Biochim Biophys Acta. 1994;1225:271-274. [Medline] [Order article via Infotrieve]
- Kaetzel DM, Maul RS, Liu B, Bonthron D, Fenstermaker RA, Coyne DW. Platelet-derived growth factor A chain gene transcription is mediated by positive and negative regulatory regions in the promoter. Biochem J. 1994;301:321-327.
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Resnick N, Collins T, Atkinson W, Bonthron DT, Dewey
CF, Gimbrone MA. Platelet-derived growth factor B chain
promoter contains a cis-acting fluid shear
stress–responsive element. Proc Natl Acad Sci U S A. 1993;90:4591-4595.
[Abstract/Free Full Text] -
Nakano T, Raines EW, Abraham JA, Wenzel FG, Higashiyama
S, Klagsbrun M, Ross R. Glucocorticoid inhibits
thrombin-induced expression of platelet-derived growth
factor A chain and heparin-binding epidermal growth
factor–like growth factor in human aortic smooth muscle
cells. J Biol Chem. 1993;268:22941-22947.
[Abstract/Free Full Text] - Zhang XY, Peng SK, Chai NN, Wan Y, Morin RJ. Influence of cholesterol oxides on gene expression of platelet-derived growth factor in cultured aortic smooth muscle cells. FASEB J. 1994;8:A49. Abstract.
- Sarria A, Moreno LA, Mur M, Lazaro A, Lasierra MP, Roda L, Giner A, Larrad L, Bueno M. Lymphocyte T subset counts in children with elevated low-density lipoprotein levels. Atherosclerosis. 1995;117:119-123. [Medline] [Order article via Infotrieve]
- Nilsson J, Svensson J, Hamsten A, de Faire U. Increased platelet-derived mitogenic activity in plasma of young patients with coronary atherosclerosis. Atherosclerosis. 1986;61:237-243. [Medline] [Order article via Infotrieve]
- Nilsson J, Volk-Jovinge S, Svensson J, Landou C, de Faire U, Hamsten A. Association between high levels of growth factors in plasma and progression of coronary atherosclerosis. J Intern Med. 1992;232:397-404. [Medline] [Order article via Infotrieve]
- Bath PMW, Martin JF. Serum platelet-derived growth factor and endothelin concentrations in human hypercholesterolemia. J Intern Med. 1991;230:313-317. [Medline] [Order article via Infotrieve]
- Cimminiello C, Arpaia G, Aloisio M, Uberti T, Rossi F, Pozzi F, Bonfardeci G. Platelet-derived growth factor in patients with different degrees of chronic arterial obstructive disease. Angiology. 1994;45:289-293.
- Ogawa H, Yasue H, Misumi I, Masuda T, Okumura K, Bannai S, Takanashi N, Tsukada Y. Plasma platelet-derived growth factor levels in coronary circulation in unstable angina pectoris. Am J Cardiol. 1992;69:453-456.[Medline] [Order article via Infotrieve]
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Wallace JMW, Turley E, Gilmore WS, Strain JJ.
Dietary fish oil supplementation alters leukocyte function and
cytokine production in healthy women.
Arterioscler Thromb Vasc Biol. 1995;15:185-189.
[Abstract/Free Full Text] -
Andersson M, Ostman A, Westermark B, Heldin C-H.
Characterization of the retention motif in the C-terminal part of the
long splice form of platelet-derived growth factor
A-chain. J Biol Chem. 1994;269:926-930.
[Abstract/Free Full Text] - Okamoto S, Inden M, Setsuda M, Konishi T, Nakano T. Effects of trapidil (triazolopyrimidine), a PDGF antagonist in preventing restenosis after percutaneous transluminal coronary angioplasty. Am Heart J. 1992;123:1439-1444. [Medline] [Order article via Infotrieve]
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Jackson CL, Raines EW, Ross R, Reidy MA. Role of
platelet-derived growth factor in arterial smooth
muscle cell migration after balloon catheter injury.
Arterioscler Thromb. 1993;13:1218-1226.
[Abstract/Free Full Text]
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