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Introduction
Elevated plasma homocysteine (Hcy) concentration is
an independent risk factor for cardiovascular
disease[1,2]. Although the exact mechanism by which
hyperhomo-cysteinemia (HHcy) induces cardiovascular disease is still
uncertain, recent evidence suggests that HHcy may induce
the process of atherogenesis by initiating inflammation. Hcy
may affect inflammatory cells, such as mononuclear
leukocytes, monocytes, and T cells, as well as multiple
cytokines, to alter the inflammatory status of endothelial cells,
smooth muscle cells (SMC) and intimal macrophages during
the formation of lesions[3,4]. Hcy induces the expression and
secretion of monocyte chemoattractant protein -1 (MCP-1)
and interleukin-8 (IL-8)in human aortic endothelial cells, SMC
lines, and monocyte cells[5-8]. Furthermore, we have shown
that Hcy not only promotes the secretion of MCP-1 and
IL-8 but also enhances their gene expression in primary
cultured human monocytes[7]. Holven
et al[9] also reported that the plasma level of the chemokine epithelial
neutrophil-activating peptide-78 (ENA-78) and growth-regulated oncogene
(GROa) was elevated in patients with HHcy. This evidence
suggests a role of pro-inflammatory chemokines in
HHcy-initiated atherogenesis.
RANTES (regulated upon activation, normal T cells
expressed and secreted) is a CC chemokine, normally derived
by T lymphocytes. It is also produced by mono/macrophages.
It plays an important role in the inflammatory process.
It has been found to play an important role in autoimmune
dis-orders such as asthma or systemic lupus erythematosus and
is nearly as potent a chemoattractant for monocytes as
MCP-1[10,11]. To date, no evidence exists on the relation
between HHcy and RANTES. In the present study, we
determined the plasma level of RANTES and the responsiveness
of monocytes to low-dose lipopolysaccharide (LPS)-induced
RANTES secretion in patients with HHcy and elucidated the
direct effect of Hcy on RANTES mRNA level in human
monocytes.
Materials and methods
Subjects Two groups of subjects were studied: 40
patients with HHcy (plasma Hcy level more than 15 µmol/L)
and 38 control patients with normal blood Hcy level (NHcy).
All subjects had no infectious diseases, malignancies or
immunologic or hematological diseases; were not being treated
with anti-inflammatory drugs other than low-dose aspirin;
did not use any vitamins including folic acid during the study
period; had no depressed cardiac function (ejection fraction
<40%); were younger than 75 years; had no renal failure;
and had not had acute myocardial infarction in the previous
3 months.
Venous blood samples were obtained from fasting
subjects to assay the chemokines from plasma and isolated
monocytes stimulated by low-dose LPS. All subjects gave their
written, informed consent. This study was approved by the
Ethics Committee of the Health Science Center, Peking
University.
Measurement of RANTES The supernatant of cultured
monocytes and plasma stored at -70 °C were used to
measure the chemokines. The concentration of RANTES was
determined by ELISA according to the manufacturer¡¯s
protocols (R&D Systems, Minneapolis, MN, USA).
Measurement of plasma level of Hcy and folate
A plasmid-encoding human placental AdoHcy hydrolase
(pPROK-1) kindly provided by the University of Kansas (USA) was
overexpressed in E coli JM109 cells transformed with
pPROK-1 and purified as described in a previous
study[12]. Total Hcy level in plasma was determined with the use of an
assay based on the conversion of Hcy to
S-adenosyl-homo-cysteine (SAH) in the presence of adenosine and
SAH hydrolase as described from our previous
study[8]. Briefly, plasma samples from fasting subjects were collected.
Protein-bound circulating Hcy was reduced to free Hcy with
the use of dithioerythreitol as a reductant. The free Hcy was
then converted to SAH by using SAH hydrolase and excess
adenosine. Samples were quantified by HPLC. The range of
measurement was 1-100 µmol/L, with a sensitivity of less
than 0.5 µmol/L. The within-assay and between-assay
coefficients of variation were 3.5% and 4.9%, respectively.
Plasma folate was measured by radioisotope dilution
assay with use of a kit from Nanjing Jiangcheng
Bioengineering Institute (Nanjing, China).
Responsiveness of human monocytes to LPS
Blood of HHcy and NHcy patients was drawn into heparinized
syringes. The whole blood was separated into peripheral
blood mononuclear cells and neutrophils by use of the
density gradient from Nycoprep 1.077 (Life Technologies, Grand
Island, NY, USA). Monocytes were then isolated from the
peripheral blood mononuclear cells by their adherence to a
serum-coated culture flask for 2 h. Adherent cells were then
detached and resuspended in RPMI-1640 medium
containing 5% autologous plasma. Freshly isolated monocytes
(5×105) were incubated at 37 ºC and treated with or without
LPS (final concentration 0.01-0.1 µg/mL) for 24 h. The
cell-free supernatant was harvested and stored at -70 ºC for
chemokine analysis. Cellular viability was determined with
Trypan blue exclusion. Only cell preparations with 95%
viability or greater were used.
RNase protection assays Total RNA was isolated from
healthy human monocytes with Trizol Reagent (Life Technologies). Assays involved use of a nuclease
protection assay kit (Riboquant, Pharmingen, San Diego, CA, USA)
as used in our previous published
report[7]. In summary, the isolated RNA (2
mg) was hybridized with 32P end-labeled
RANTES oligonucleotide probes overnight at 30
°C, followed by nuclease digestion. A GAPDH and L-32 rRNA
oligonucleotide probe was used as an internal control. After
digestion, the protected fragments were resolved on a
denatured 12% polyacrylamide gel containing 8 mol/L urea, then
transferred to filter paper, which was later exposed to X-ray
film. The bands corresponding to RANTES, L-32 or GAPDH
mRNA were analyzed by use of a gel documentation system
(Cold-Spring Eletro Doc and Analyst Edas 290, New York,
USA).
Statistical analysis Because chemokine and Hcy values
do not follow a normal distribution in patients, comparisons
between groups involved the use of the Mann-Whitney test.
Values are expressed as median, 25th and 75th percentile,
and 10th and 90th percentile. Patients¡¯ age, body-mass
index and plasma level of cholesterol, triglycerides and
creatinine were analyzed with use of the Student¡¯s
t-test and expressed as mean±SD. Proportions were analyzed by use of
the Chi-squared test. P<0.05 (two-tailed) was considered
statistically significant.
Results
Clinical characteristics of the study participants
The NHcy control and HHcy groups did not differ significantly
with respect to age, sex, and body-mass index. The
prevalence of smoking, coronary artery disease, hypertension and
diabetes, as well as base-line demographic characteristics
and laboratory values, were also similar in the two groups
(Table 1).
Plasma level of Hcy, folate, and RANTES The Hcy level
was significantly higher in HHcy subjects than that in NHcy
controls, while the folate level was lower in HHcy subjects
than that in NHcy controls. In the HHcy group, the plasma
level of RANTES was significantly elevated (median 5.3
ng/mL) compared with NHcy controls (3.5 ng/mL) (Table
2).
Chemokine secretion from isolated monocytes in
response to LPS To test whether monocytes isolated from
patients with HHcy exhibited enhanced inflammatory
response of RANTES production, monocytes were incubated
with low-dose LPS (0.01 µg/mL) for 24 h. As shown in Figure
1, RANTES production was not significantly increased in HHcy
patients without LPS stimulation, but LPS treatment (0.01
µg/mL) significantly increased the level of RANTES
secretion of monocytes from HHcy patients compared with that
of NHcy controls. Thus, monocytes from patients with HHcy
may show an enhanced RANTES secretion response in a
pro-inflammatory condition.
Effect of Hcy on RANTES mRNA expression To
determine whether Hcy modulates the expression of RANTES
mRNA, total RNA was isolated from normal human
monocytes treated with Hcy at various times and at different
concentrations. As shown in Figure 2, RNase protection
assays revealed that the expression of RANTES mRNA was
significantly enhanced after Hcy treatment. Cultured
monocytes incubated with Hcy (10-1000 µmol/L) for 8 h showed
significantly increased RANTES expression as compared with
untreated cells (Figure 2A), beginning as low as Hcy 10
µmol/L. The increased level of RANTES mRNA after incubation with
Hcy 100 mmol/L peaked at 4-8 h after incubation (Figure 2B).
Discussion
The results from the present study demonstrated that
the plasma RANTES level was elevated in HHcy individuals
and RANTES secretion from monocytes was increased. We
also provided evidence that Hcy directly induced RANTES
mRNA synthesis from isolated human monocytes.
Atherosclerosis is a chronic inflammatory disease
characterized by the recruitment of monocytes and lymphocytes
to the artery wall. Increasing evidence supports the
involvement of inflammation in the early phases of atherogenesis.
Recruitment of leukocytes within the vascular wall is an
essential process in the development of this common disease,
which is mainly regulated by adhesion to molecules and
chemokines[3,4]. In the classic inflammatory response,
adhesion is followed by transmigration of the leukocytes through
the endothelial layer into the intima. This process is also
mediated by chemotactic factors.
A number of studies have determined the important role
of chemokines such as MCP-1 and IL-8 in the initial stages
of atherosclerotic plaque
formation[5,6]. RANTES, a CC chemokine, has been found to play an important role in
autoimmune injury to several tissues. RANTES is generated
by circulatory lymphocytes and some kinds of tissue cell
monocytes. RANTES is nearly as potent a chemoattractant
for monocytes as MCP-1. RANTES induces leukocyte transendothelial migration, implicated in the initial stages of
the inflammatory part of the atherosclerotic
process[13]. Furthermore, evidence implies the important role RANTES
plays in atherosclerosis. Simeoni et
al[14] reported that a mutant RANTES genotype was associated with the increased
coronary heart disease death rate, independent of
conventional risk factors. A causal role for RANTES in
atherosclerosis was also shown by a protective effect of the blockage
of RANTES receptors with the CC chemokine antagonist
Met-RANTES in an ApoE-deficient hypercholesterolemic
mouse model[15]. These data suggest that the alteration of
the level of the chemokine RANTES may affect the progress
of atherogenesis through the inflammatory pathway.
Hyperhomocysteinemia is found in 30% of patients with
premature atherosclerosis of carotid and peripheral arteries.
Elevated plasma Hcy levels have been implicated as an
independent risk factor for coronary heart
disease[16,17]. Therefore, intensive studies have focused on whether HHcy is the cause
or merely a marker for cardiovascular disease. Hcy
significantly enhances MCP-1 and IL-8 levels in healthy human
monocytes, which can increase leukocyte
chemotaxis[7]. However, compared with studies of MCP-1 and IL-8, little
study has focused on RANTES secretion in HHcy. Economou
et al[17] reported the negative association of
circulating Hcy and RANTES in prepubescent lean children.
Until now, no direct evidence of the relation between HHcy
and RANTES in human monocytes exists.
In the present study, we found that both the plasma level
of RANTES and low-dose LPS-induced RANTES production in isolated monocytes from patients with HHcy were
significantly elevated. Hcy not only increased the RANTES
level in HHcy patients but also increased RANTES gene
expression in primary human monocytes. Thus, our results
support our hypothesis that HHcy may play an important
role in the pathogenesis of atherosclerosis through a
mechanism involving an increase in RANTES secretion in human
monocytes.
Our recent published data show that folate acid
treatment in HHcy patients can reverse the
hyper-responsiveness of MCP-1 and IL-8 secretion from
monocytes[8]. On the basis of the current results, further study should focus on
whether the intervention of HHcy influences
monocyte-derived RANTES secretion and also the exact mechanism
underlying the Hcy-upregulated RANTES production.
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