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Third Department of Internal Medicine, Kurume University School of Medicine, Kurume, Japan.
Correspondence to Dr. Masahisa Fujisawa, Third Department of Internal Medicine, Kurume University School of Medicine, 67 Asahi-machi, Kurume 830-0011, Japan. Phone: +81-942-31-7562; Fax: +81-942-33-6509; E-mail: fujisawa{at}med.kurume-u.ac.jp
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A number of factors, including age, hypertension, hyperlipidemia, and diabetes, have been found to be strongly associated with an increased incidence of atherosclerosis in the general population. Some of these conditions have a higher prevalence in patients with CRF, which may explain the occurrence of accelerated atherosclerosis in HD patients. Among them, dyslipidemia has been focused on as an accelerating factor for atherosclerosis. Uremic patients have a unique lipoprotein profile called uremic dyslipidemia (4), which is characterized by hypertriglyceridemia (5), elevated very-low-density lipoprotein VLDL, accumulated intermediate-density lipoprotein, and decreased high-density lipoprotein (HDL) (6, 7). Especially, lipoprotein (a) [Lp(a)] has been shown to be elevated in CRF (8, 9) and to be related to atherogenesis through the inhibition of transforming growth factor-ßa (TGF-ß1) (10, 11).
Recent evidence obtained through experiments done in vitro suggests that TGF-ß1 may regulate progression of atherosclerosis (10, 11). Atherosclerotic lesions are thought to originate from injury or dysfunction of the endothelium (12). In response to various agents acting at the site of injury, including plateletderived growth factor (PDGF) and other mitogens, the underlying vascular smooth muscle cells (VSMC) migrate into the lumen and proliferate to form an intima (13). In contrast, TGF-ß1 inhibits both migration (14) and proliferation (10) of VSMC in vitro. TGF-ß1 is usually produced in a latent, inactive form, which is activated proteolytically by plasmin, a serine protease (15,16,17). Plasmin is produced proteolytically from plasminogen activator on the cells (15), and the risk factor Lp(a) blocks the activation of latent TGF-ß1 by competitively inhibiting plasminogen activator. Lp(a), therefore, promotes VSMC proliferation by relieving the autocrine inhibition caused by active TGF-ß1 (10).
The component of Lp(a) that acts as an inhibitor of plasminogen activator is apolipoprotein (a) [apo(a)], which has 80% amino acid sequence homology with the corresponding domains in plasminogen (18). Evidence obtained from experiments in a transgenic mouse model of atherosclerosis in which human apo(a) is expressed supports the idea of a close correlation between accumulation of apo(a) on the vessel wall and inhibition of TGF-ß1 activation (11). At the site of apo(a) accumulation, the VSMC are activated and vascular lesions subsequently develop. Thus, active TGF-ß1 may be a key inhibitor of atherogenesis.
In this study, we investigated the concentration of total TGF-ß1 and mature TGF-ß1 in circulation and in culture supernatants of peripheral mononuclear cells from HD patients before and after an HD session. Then, the TGF-ß1 data were analyzed to clarify the effects of Lp(a) on TGF-ß1 activation and the influence of TGF-ß1 on atherosclerosis in HD patients.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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- or
ß-blockers, oral nitrates, and diuretics. Blood samples were collected
under fasting conditions. Plasma lipid levels were determined in an
autoanalyzer (HR2400, Nihon-Denshi, Tokyo) by standard enzymatic methods.
Plasma Lp(a) levels were measured by a latex immunoturbidimetric assay
(19). The intra-assay
variation coefficient of Lp(a) concentration was a mean of 6.9%.
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Blood Sampling and Mononuclear Cell Culture
Peripheral venous blood was sampled for the assay of plasma TGF-ß1 and
for mononuclear cells culture. The differential counts of white blood cells
were performed in every patient. The ratio of monocytes/lymphocytes was
relatively constant (31.0 ± 15.3%). Preand postdialysis blood samples
were obtained on the same day. Blood was collected into a sample tube
containing EDTA and centrifuged. Plasma was stored at - 70°C until
assayed. Mononuclear cells were separated by Ficoll gradient centrifugation
and suspended in RPMI 1640 medium
(20). Cells seeded at a cell
density of 2 x 106/ml were incubated for 24 h in
flat-bottomed six-well culture plates (3 ml/well; Nunc, Paskide, Denmark).
Supernatants were collected and stored at -70°C.
TGF-ß1 Assay
Mature and total (mature + latent) TGF-ß1 levels in plasma and in the
supernatant of mononuclear cells culture were measured with the use of a
TGF-ß1-specific sandwich enzyme-linked immunosorbent assay (ELISA;
Promega, Madison, WI). This quantitative sandwich enzyme immunoassay was based
on an immunomobilized monoclonal antibody, which detects only unbound mature
TGF-ß1. Plasma and supernatant from the mononuclear cells culture were
treated with acid to dissociated TGF-ß1 complexes because TGF-ß1 in
latent complexes is not recognized by antibodies directed against the mature
form of TGF-ß1 (21).
Briefly, 1 N HCl was added to plasma and supernatant of cultured mononuclear
cells; these were allowed to stand for 15 min at room temperature to lower the
pH to 2.6, and then they were neutralized with 1 N NaOH to a pH of 7.4. Each
sample of plasma and supernatant of mononuclear cells was diluted to 1:300 and
1:5, respectively, in TGF-ß1 sample buffer and added to the ELISA plate.
To measure the amount of mature TGF-ß1, we added the samples directly
into the ELISA plate after being diluted in TGF-ß1 sample buffer (1:5
[vol:vol] in case of plasma and 1:1 [vol:vol] in that of supernatant). A
TGF-ß1 standard curve was produced by a twofold serial dilution with
final concentrations of 1000, 500, 250, 125, 62.5, 31.25, and 15.6 pg/ml using
a recombinant human TGF-ß1 standard. Absorbance was measured at a
wavelength of 450 nm on a plate reader (Bio-Rad model 550, Hercules, CA) to
determine TGF-ß1 concentration, then a curve-fitting software program was
used to quantify the TGF-ß1 concentration in the samples. The minimum
level of detection was 25 pg/ml of TGF-ß1. For reproducibility analysis,
10 different plasma samples were repeatedly measured on three separate
occasions. The intra-assay variation were 1.5 to 20.5% with a mean variation
of 8.5%. The intra-assay variation for nine samples analyzed in triplicate was
0.25 to 2.53% with a mean of 1.5%.
Ultrasonographic Assessment of Carotid Arteries
Ultrasonographic scanning of the carotid artery was performed with the use
of a high-resolution ultrasonographer (SSA-270A, Toshiba, Tokyo) provided with
an 8.0-MHz transducer. Each subject was examined in the supine position in a
semidark room. The carotid artery was investigated bilaterally. The carotid
artery was scanned at the level of the bifurcation of the common carotid
arteries. IMT was taken as the distance from the leading edge of the first
echogenic line to the leading edge of the second echogenic line. IMT was
measured on the longitudinal views of the far wall of the bilateral distal
common carotid arteries (1 to 3 cm proximal to the carotid bifurcation) at the
diastolic phase. Then IMT was expressed as the mean of six measurements (three
on each side) (22). In case of
lesions calcified as a plaque, a focal calcified hyperechogenic thickening of
more than 1 mm with a distal hypoechogenic zone was displayed. Plaque
thickness was measured in a suitable longitudinal view or transverse view. The
plaque score was calculated by summing up the thickness of all of the plaques
for both carotid systems
(23).
Statistical Analyses
The results are presented as means ± SEM. We used analysis of
variance when the three groups were compared. We also used paired t
test to analyze the distribution of TGF-ß1 before and after an HD
session. Correlations were analyzed by linear regression analysis, and the
coefficient of correlation was determined. A P value of less than
0.05 was considered statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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IMT and Plaque Score in HD Patients
To evaluate the degree of atherosclerosis in HD patients, we measured IMT
and plaque score in the carotid arteries, using an ultrasound scanner. IMT and
plaque score were significantly increased in non-HD-CRF and HD patients than
in control subjects (Figure 2, A and
B). Then IMT and plaque score were compared with risk factors for
atherosclerosis, such as age, Lp(a), total cholesterol, triglycerides, LDL
cholesterol, HDL cholesterol, BP, and HD duration. A significant positive
correlation was found between IMT and age
(Figure 3A; r = 0.800,
P < 0.001), between IMT and Lp(a)
(Figure 3B; r = 0.337,
P < 0.01), between plaque score and age
(Figure 4A; r = 0.55,
P < 0.001, and between plaque score and Lp(a)
(Figure 4B; r = 0.43,
P < 0.01) in HD patients. However, neither IMT nor plaque score
showed any significant correlation with the other risk factors such as total
cholesterol, triglycerides, LDL cholesterol, BP, or HD duration in HD
patients. These results indicated that age and Lp(a) were strongly associated
with atherosclerosis in HD patients.
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Plasma TGF-ß1
The plasma level of total TGF-ß1 was 17.70 ± 1.77 ng/ml in the
control subjects, which was similar to the value reported previously
(24). Total TGF-ß1 was
significantly increased in HD patients and non-HD-CRF patients compared with
control subjects (Figure 5A).
There was no significant difference in plasma total TGF-ß1 between HD and
non-HD-CRF patients. The plasma level of mature TGF-ß1 was 0.378 ±
0.031 ng/ml, which indicated a 0.36% activation rate of TGF-ß1 in plasma
in HD patients. There were no significant differences in the plasma level of
mature TGF-ß1 among the groups (Figure
5B). The activation rate of TGF-ß1 (ratio of mature
TGF-ß1/total TGF-ß1) was significantly lower in HD and non-HD-CRF
patients than in control subjects (Figure
5C). These results revealed an increase in plasma total
TGF-ß1 and a decrease in the activation rate in CRF patients.
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TGF-ß1 Produced by Cultured Peripheral Mononuclear Cells
Culture supernatant from cultured mononuclear cells contained 2.08 ±
0.13 ng/ml of total TGF-ß1 in the HD group. There were no significant
differences in the concentration of total TGF-ß1 in culture supernatant
among the HD, non-HD-CRF, and control groups
(Table 2). The concentration of
mature TGF-ß1 in culture supernatant was 0.23 ± 0.16 ng/ml, which
indicated 13% activation rate of TGF-ß1 in HD patients. No significant
differences were found regarding the concentration of mature TGF-ß1 in
culture supernatant from mononuclear cells among the three groups
(Table 2).
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Effects of a Dialysis Session on TGF-ß1 Production and
Activation
To investigate whether HD stimulated the production or activation of
TGF-ß1 in HD patients, we compared the concentrations of total and mature
TGF-ß1 in plasma and in culture supernatants from mononuclear cells
before and after an HD session. The concentrations of plasma total and mature
TGF-ß1 were corrected by the hematocrit before and after HD. Plasma
concentrations of total TGF-ß1 and mature TGF-ß1 before HD were
141.9 ± 10.2 ng/ml and 0.41 ± 0.05 ng/ml, respectively. These
values were not different from those of total TGF-ß1 and mature
TGF-ß1 after HD session, 143.2 ± 9.9 ng/ml and 0.45 ± 0.06
ng/ml. The concentrations of total TGF-ß1 and mature TGF-ß1 in
culture supernatants of peripheral mononuclear cells were 2.02 ± 0.15
ng/ml and 0.23 ± 0.01 ng/ml, respectively. These values were not
different from those of total TGF-ß1 and mature TGF-ß1 after HD
session, 1.83 ± 0.18 ng/ml and 0.21 ± 0.02 ng/ml.
Plasma TGF-ß1 and Atherosclerosis in HD Patients
To examine the influence of TGF-ß1 on atherosclerosis in HD patients,
we evaluated the correlations between plasma TGF-ß1 and IMT or plaque
score. There was no significant correlation between the plasma total
TGF-ß1 and IMT (r = 0.02, P = 0.89) between the plasma
mature TGF-ß1 and IMT (r = -0.01, P = 0.50), between
the plasma total TGF-ß1 and plaque score (r = -0.01, P
= 0.94), or between the plasma mature TGF-ß1 and plaque score in HD
patients (r = 0.05, P = 0.74).
Mononuclear Cell TGF-ß1 Production and Atherosclerosis in HD
Patients
To examine the participation of mononuclear TGF-ß1 production in the
development of atherosclerosis in HD patients, we evaluated the correlations
between total and mature TGF-ß1 in culture supernatant of mononuclear
cells and IMT or plaque score. There was no significant correlation between
the supernatant total TGF-ß1 and IMT (r = -0.20, P =
0.17), between the supernatant mature TGF-ß1 and IMT (r = 0.05,
P = 0.75), between the supernatant total TGF-ß1 and plaque score
(r = -0.25, P = 0.08), or between the supernatant mature
TGF-ß1 and plaque score in HD patients (r = -0.04, P =
0.78).
Plasma TGF-ß1 and Lp(a) in HD Patients
To investigate the inhibitory effects of Lp(a) on TGF-ß1 activation,
we compared the plasma levels of total and mature TGF-ß1 with the plasma
level of Lp(a) in HD patients. The Lp(a) level showed no correlation with
total TGF-ß1 (r = -0.05, P = 0.71), with mature
TGF-ß1 (r = 0.10, P = 0.48), or with the activation
rate of TGF-ß1 (mature/total TGF-ß1; r = 0.03, P =
0.86).
Mononuclear Cell TGF-ß1 Production and Lp(a) in HD)
Patients
To investigate the inhibitory effects of Lp(a) on TGF-ß1 production of
mononuclear cells, we compared the TGF-ß1 level in culture supernatant of
mononuclear cells with that of Lp(a) in HD patients. The Lp(a) level showed no
correlation with total TGF-ß1 (r = 0.02, P = 0.91),
with mature TGF-ß1 (r = -0.06, P = 0.67), or with the
activation rate of TGF-ß1 (mature/total TGF-ß1; r = 0.01,
P = 0.93) of culture supernatant of mononuclear cells in HD
patients.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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HD has been reported to stimulate cytokine production (25,26,27,28,29). It has been thought that dialysis activates monocytes and macrophages, leading to the release of cytokines (30). TGF-ß1 released by immune cells, such as activated T cells and monocytes/macrophages (31, 32), may exert a variety of activities after a dialysis session. Mege and colleagues (32) reported that TGF-ß1 and TGF-ß2 production by peripheral monocytes was significantly higher in HD patients than in control subjects. In the present study, total TGF-ß1 was also increased in HD patients compared with healthy control subjects. However, the plasma concentration of TGF-ß1 in HD patients was similar to that in non-HD-CRF patients. In addition, when plasma TGF-ß1 and TGF-ß1 in the culture supernatant of mononuclear cells isolated after an HD session were compared with those before the session, no significant increases in TGF-ß1 were detected. These results suggest that the increased plasma concentration of total TGF-ß1 observed in HD patients is unlikely to be due to a release from peripheral mononuclear cells activated by the dialyzer membrane. The most probable mechanism is a reduced renal TGF-ß1 degradation. As renal insufficiency progresses, the tubular and peritubular uptake of polypeptides decreases, causing a disproportionate rise in serum concentrations. This reduced renal peptide degradation may be responsible for the increased concentration of total TGF-ß1 in CRF.
Atherosclerotic vascular disease is a major cause of death in uremic patients who are on HD, and they have an increased intima-media thickness of the carotid and femoral arteries. Dyslipidemia is common among patients with CRF (4,5,6,7,8,9). A recent study showed that HD patients with atherosclerotic events had Lp(a) levels twice as high as those of HD patients without events (33). In the present study, plasma Lp(a) increased in CRF patients independent of HD and showed a close correlation with IMT and plaque score, suggesting the crucial role of Lp(a) in atherogenesis in CRF patients.
A component of Lp(a) that acts as an inhibitor of plasminogen activator has been reported to inhibit the activation of TGF-ß1 (10). Evidence from a transgenic mouse model of atherosclerosis in which human apo(a) is expressed provides support for a close correlation between accumulation of apo(a) on the vessel walls and inhibition of TGF-ß1 activation. At the site of atherosclerosis, VSMC migrate into the lumen and proliferate to form an intima (13) in response to various agents acting at the site of injury, including PDGF and other mitogens, and vascular lesions subsequently develop. TGF-ß1 may regulate progression of atherosclerosis by inhibiting both migration (14) and proliferation (10) of VSMC. TGF-ß1 is activated proteolytically by the serine protease plasmin (15,16,17). Lp(a), therefore, may promote human VSMC proliferation in culture by relieving the autocrine inhibition caused by active TGF-ß1 (10), because Lp(a) blocks the activation of latent TGF-ß1 by competitively inhibiting the plasminogen activator.
Grainger and colleagues
(34) reported that in a group
of patients with advanced atherosclerosis, all of them had lower levels of
mature TGF-ß1 in their sera than did patients with normal arteries. They
suggested that mature TGF-ß1 had a diagnostic and prognostic significance
and might be a key inhibitor of atherogenesis. In the present study, the
activation rate of TGF-ß1 (mature/total TGF-ß1) was significantly
reduced in HD and non-HD-CRF patients. Although Lp(a) was increased in HD
patients compared with normal control subjects and correlated with IMT and
plaque score, there was no significant correlation between plasma Lp(a) and
plasma mature TGF-ß1 or the activation rate of TGF-ß1. In addition,
there was actually no difference in total or mature TGF-ß1 between 10
nonuremic coronary artery disease patients and 13 healthy subjects in our
field study, although the number of subjects was small (data not shown). These
results suggest that TGF-ß1 might not show any inhibitory or contributory
effects on atherogenesis in HD patients and that Lp(a) may work as an
atherogenic factor independent of TGF-ß1 in HD patients. However, it is
unclear whether the rate of plasma mature/total TGF-ß1 represents an
activation process in CRF because mature TGF-ß1 is rapidly cleared from
the circulation. In studies on the metabolism of TGF-ß1, mature
TGF-ß1 disappeared rapidly from the circulation by 2-macroglobulin
binding or by hepatic processing
(35,
36). Furthermore, there is a
possibility that mature TGF-ß1 may be removed through the dialysis
membrane instead of by intradialysis activation of TGF-ß1 in HD patients
because the molecular size of mature TGF-ß1 (25 kD) is smaller than the
transfer limitation of high-flux dialysis membranes. In contrast, the latent
TGF-ß1 complex has a large molecular size (105 to 310 kD) and a longer
plasma half-life than active TGF-ß1 and is distributed to a variety of
organs, including the kidneys
(37). Furthermore, the lower
ratio of plasma mature/total TGF-ß1 may be due to the increased
concentration of latent TGF-ß1, which remained in the plasma of CRF
patients. The differences in half-life between latent TGF-ß1 and mature
TGF-ß1 may explain why the plasma concentration of mature TGF-ß1 was
not affected by renal function. Thus, the present study did not support the
hypothesis that Lp(a) may exert atherogenic effects by inhibiting TGF-ß1
activation carried in HD patients.
In summary, the degree of atherosclerosis was more advanced in CRF patients (HD and non-HD-CRF) compared with healthy control subjects. Plasma Lp(a) was significantly correlated with the degree of atherosclerosis in CRF patients. Plasma total TGF-ß1 was increased in CRF patients, whereas total TGF-ß1 or TGF-ß1 activation rate showed no significant correlation with plasma Lp(a) or the degree of atherosclerosis. It was concluded that Lp(a) may be an important atherogenic factor in CRF patients. However, it was not clarified whether Lp(a) exerts its atherogenic effects by inhibiting TGF-ß1 activation in CRF patients.
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Acknowledgments |
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