A possible association between cholesterol levels and inflammatory markers, such as oxidized low-density lipoprotein, highly sensitive C-reactive protein and oxidized low-density lipoprot
Trang 1with hypercholesterolemia and inflammatory processes Marta Medeiros Frescura Duarte1, Vaˆnia L Loro1, Joa˜o B T Rocha1, Daniela B R Leal2,
Andreza F de Bem1, Arace´li Dorneles1, Vera M Morsch1 and Maria R C Schetinger1
1 Departamento de Quı´mica, Centro de Cieˆncias Naturais e Exatas, Programa de Pos-Graduac¸a˜o em Bioquı´mica Toxicolo´gica, Universidade Federal de Santa Maria, Brazil
2 Hospital Universita´rio de Santa Maria, Santa Maria, RS, Brazil
Hypercholesterolemia is widely accepted as one of the
major risk factors for the development of ischemic heart
disease, angina, and myocardial infarction Although
the risks imposed by hypercholesterolemia seem to be
manifold, most attention has been devoted to its role in
atherosclerosis [1] Atherosclerosis is an inflammatory
disease that is associated with endothelial cell activation,
oxidative stress, and the accumulation of leukocytes in
the walls of arteries [2] The inflammatory process
induced by hypercholesterolemia is not limited to large arteries Endothelial cell adhesion molecule expression, enhanced oxidant production and leukocyte–endothelial cell adhesion have been demonstrated in postcapillary venules of different tissues of hypercholesterolemic animals [3–6]
Low-density lipoprotein is a major carrier of cholesterol in the circulation, and can play an impor-tant role in atherogenesis if it undergoes oxidative
Keywords
cholesterol; highly sensitive C-reactive
protein; NTPDase; oxidized low-density
lipoprotein; oxidized low-density lipoprotein
autoantibodies
Correspondence
M R C Schetinger, Departamento de
Quı´mica, Centro de Cieˆncias Naturais e
Exatas, Universidade Federal de Santa
Maria, Av Roraima 1000, 97105-900, Santa
Maria, RS, Brazil
Fax: +55 5532 208978
Tel: +55 5532 208665
E-mail: mariaschetinger@gmail.com
(Received 18 August 2006, revised 29
January 2007, accepted 19 March 2007)
doi:10.1111/j.1742-4658.2007.05805.x
The activity of NTPDase (EC 3.6.1.5, apyrase, CD39) was verified in plate-lets from patients with increasing cholesterol levels A possible association between cholesterol levels and inflammatory markers, such as oxidized low-density lipoprotein, highly sensitive C-reactive protein and oxidized low-density lipoprotein autoantibodies, was also investigated Lipid per-oxidation was estimated by measurement of thiobarbituric acid reactive substances in serum The following groups were studied: group I,
< 150 mgÆdL)1cholesterol; group II, 151–200 mgÆdL)1 cholesterol; group -III, 201–250 mgÆdL)1 cholesterol; and group IV, > 251 mgÆdL)1 choles-terol The results demonstrated that both ATP hydrolysis and ADP hydrolysis were enhanced as a function of cholesterol level Low-density lipoprotein levels increased concomitantly with total cholesterol levels Tri-glyceride levels were increased in the groups with total cholesterol above
251 mgÆdL)1 Oxidized low-density lipoprotein levels were elevated in groups II, III, and IV Highly sensitive C-reactive protein was elevated in the group with cholesterol levels higher than 251 mgÆdL)1 Oxidized low-density lipoprotein autoantibodies were elevated in groups III and IV Thiobarbituric acid reactive substance content was enhanced as a function
of cholesterol level In summary, hypercholesterolemia is associated with enhancement of inflammatory response, oxidative stress, and ATP and ADP hydrolysis The increased ATP and ADP hydrolysis in group IV was confirmed by an increase in CD39 expression on its surface The increase
in CD39 activity is possibly related to a compensatory response to the inflammatory and pro-oxidative state associated with hypercholesterolemia
Abbreviations
hsCRP, highly sensitive C-reactive protein; OxLDL, oxidized low-density lipoprotein; PRP, platelet-rich plasma.
Trang 2modification by endothelial cells, vascular smooth
muscle, or macrophages within the arterial wall [7]
Oxidized low-density lipoprotein (OxLDL) as well as
OxLDL autoantibodies have been found in several
studies in atherosclerotic lesions [8] The highly
sensitive C-reactive protein (hsCRP) enhances the
binding of OxLDL to monocytic⁄ macrophage-like
cells through Fcc receptors [9]
Platelets also accumulate within atherosclerotic
lesions, and can recruit additional platelets to form a
thrombus, indicating that the arterial wall can assume
both an inflammatory and prothrombogenic phenotype
when blood cholesterol levels are elevated [10,11]
Platelets are one of the most important blood
compo-nents that participate in and regulate thrombus
forma-tion by releasing active substances, such as ADP [12]
Micromolar concentrations of ADP are sufficient to
induce human platelet aggregation, and in the
coagula-tion cascade, AMP is hydrolyzed to adenosine, which
has an important function in the regulation of platelet
aggregation [13–15] Furthermore, the roles of
nuc-leotides and nucleosides as extracellular signaling
molecules have been well established Extracellular
nu-cleotides have become recognized for the significant
role that they play in modulating a variety of processes
related to vascular inflammation and thrombosis
[16–19] In this vein, recent data from our laboratory
have indicated changes in nucleotide hydrolysis by
platelets from patients carrying diseases generally
asso-ciated with changes in coagulation⁄ homeostasis
[13–15] More recently, there has been growing interest
in the long-term effects of extracellular nucleotides and
nucleosides on cell growth, proliferation, and death
[16–19]
NTPDase (CD39, ecto-apyrase, ATP
diphospho-hydrolase) is a glycosylated, membrane-bound enzyme
that hydrolyzes ATP and ADP to AMP, which is
sub-sequently converted to adenosine by 5¢-nucleotidase
(EC 3.1.3.5, CD73) NTPDases are located in various tissues, including the platelet membrane [20,21] NTP-Dase and CD73 play an important role in the regula-tion of blood flow and thrombogenesis by regulating ADP catabolism [22] Another aspect that must be emphasized here is the fact that NTPDase1 rapidly metabolizes ADP released during platelet activation This event is very important, because ADP is the final mediator of platelet recruitment and thrombus forma-tion [23] In fact, NTPDase1 has been recognized to play an important role in thromboregulation by hydro-lyzing and lowering extracellular ADP, which inhibits platelet aggregation In a recent study, Papanikolaou
et al [24] have shown that depletion of membrane cho-lesterol results in strong inhibition of NTPDase, and that this is reversed by purified cholesterol
Thus, one question that could be asked is whether the different cholesterol levels observed in human blood could affect NTPDase1 activity This study was performed in an attempt to answer this question The activity of NTPDase1 was measured on platelets of human donors with cholesterol levels ranging from less than 150 to more than 251 mgÆdL)1 Furthermore,
we studied whether cholesterol levels were associated with inflammatory markers, e.g hsCRP, and with lipid peroxidation
Results Patient characteristics are shown in Table 1 Glucose levels were in the normal range in all the groups studied, and ranged from 88 to 99 mgÆdL)1 No signi-ficant differences were observed in the high-density lipoprotein cholesterol levels among the groups Conversely, low-density lipoprotein levels increased concomitantly with the increase in total cholesterol levels, and were significantly higher in groups III and
IV Triglyceride levels were increased in group IV (the
Table 1 Characteristics of the four groups: age (years), sex (male ⁄ female), smoking, hypolipemic medication, and anti-inflammatory treat-ment Age is represented by mean ± SE.
Patients
Cholesterol groups
I (< 150 mgÆdL)1) II (151–200 mgÆdL)1) III (201–250 mgÆdL)1) IV (> 251 mgÆdL)1)
Sex
Hypolipemic
medication
Anti-inflammatory
treatment
Trang 3group with total cholesterol above 251 mgÆdL)1;
Table 2)
OxLDL was elevated in groups II, III, and IV
(Table 3), whereas hsCRP was elevated only in
group IV (cholesterol higher than 251 mgÆdL)1)
Ox-LDL autoantibodies were elevated in groups III and
IV (Table 3)
A statistically significant positive correlation was
found between the cholesterol levels and thiobarbituric
acid reactive substance production (r¼ 0.7438,
P< 0.01), which indicates an association between
plasma⁄ serum ⁄ blood and oxidative stress (Fig 1)
ATP hydrolysis was modified by cholesterol levels
above 151 mgÆL)1, and post hoc comparisons by
Duncan’s test revealed that it was significantly higher
in patients from groups II, III, and IV Furthermore,
these groups were significantly different from each
other (Fig 2A) Similar results were observed for
ADP hydrolysis Post hoc comparisons by Duncan’s
multiple range test revealed that patients from
groups II, III and IV presented higher levels of ADP
hydrolysis and that these groups were significantly
different from each other (Fig 2B) Correlation
ana-lysis indicated a positive correlation between
increased cholesterol levels and platelet ATP and
ADP hydrolysis (Fig 3A,3B)
Statistical analysis of the content of CD39-positive
cells by flow cytometry using labeled antibody against
NTPDase1 revealed that group IV (cholesterol higher
than 251 mgÆdL)1) had a significant increase in the
expression of NTPDase1, when compared to the other groups (P < 0.05, post hoc comparisons by Duncan multiple test; Fig 4)
There was a statistically significant correlation between ATP and ADP hydrolysis with OxLDL (r¼ 0.82, P < 0.01; r ¼ 0.91, P < 0.001), hsCRP (r¼ 0.82, P< 0.01; r¼ 0.91, P < 0.001), and OxLDL autoantibodies (r¼ 0.85, P < 0.01; r ¼ 0.96,
P < 0.001) ATP and ADP hydrolysis were also corre-lated with tryglyceride levels (r¼ 0.819, P < 0.001;
r ¼ 0.92, P < 0.001; Table 4)
Table 2 High-density lipoprotein (HDL), low-density lipoprotein (LDL), triglyceride and glucose (mg⁄ dL) levels of patients with different cho-lesterol levels Results are expressed as the mean ± SE (n ¼ 40 for each group).
Blood parameters
Cholesterol groups
I (< 150 mgÆdL)1) II (151–200 mgÆdL)1) III (201–250 mgÆdL)1) IV (> 251 mgÆdL)1)
* Indicates significant difference at P < 0.05 between groups.
Table 3 OxLDL (mg ⁄ dL), hsCRP (mg ⁄ L) and OxLDL autoantibodies (mg ⁄ L) in patients with different cholesterol levels Results are expressed as the mean ± SE (n ¼ 40 for each group).
Parameters
Cholesterol levels
I (< 150 mgÆdL)1) II (151–200 mgÆdL)1) III (201–250 mgÆdL)1) IV (> 251 mgÆdL)1)
* Indicates significant difference at P < 0.05 between groups.
35 30 25 20 15 10
Cholesterol (mg/dl)
5 0
0 50 100 150 200 250 300 350 400 450
Fig 1 Correlation between cholesterol levels and thiobarbituric acid reactive substances (n ¼ 40) (r ¼ 0.74, P < 0.01) y ¼ 4.37 + 0.077x, where y ¼ malondialdehyde production (nmolÆmL)1) and x ¼ cholesterol levels.
Trang 4The present study clearly indicated that cholesterol
lev-els are associated with increased OxLDL, OxLDL
autoantibody formation, and inflammatory markers
The results of this study reveal that patients with high
cholesterol may have a predisposition to atheroma
pla-que development The results of the present study also
show a positive correlation between cholesterol levels
and OxLDL and hsCRP production, which is in
accordance with the literature data [7] Our study
con-firmed the hypothesis that increased levels of
choles-terol may be associated with hsCRP and OxLDL
autoantibodies, which are good indicators of increased
risk for atherosclerosis development
The present results show for the first time that
nucleo-tide hydrolysis is enhanced in platelets of patients
with hypercholesterolemia Probably, high cholesterol
levels induce an increase in platelet ATP and ADP
hydrolysis as a compensatory mechanism to inhibit
platelet aggregation and limit thrombus formation in
patients with a predisposition to atheroma development
30
A
B
< 150
> 251
*
*
*
*
*
*
201-250 151-200
< 150
> 251 201-250 151-200
20
10
0
10.0
7.5
5.0
2.5
0.0
< 150
151-200 Cholesterol (mg/dl)
201-250 > 251
< 150 151-200
Cholesterol (mg/dl)
201-250 > 251
Fig 2 ATP (A) and ADP (B) hydrolysis in platelets from patients
with hypercholesterolemia (n ¼ 40) Results are expressed as
nmol P i Æmin)1Æmg)1of protein Different letters indicate a significant
difference at P < 0.05 between groups.
A 40
30
20
10
0
0 50 100 150 200
Cholesterol (mg/dl)
250 300 350 400
80
10
8
6
4
2
0
120 160 200 240 280 320 360 400
450
B
Fig 3 Correlation analysis between cholesterol levels and ATP (A) and ADP (B) hydrolysis (n ¼ 40) (r ¼ 0.75, P < 0.01) y ¼ 2.06 + 0.078x, where y ¼ ATP hydrolysis and x ¼ cholesterol levels.
< 150
> 251
*
201-250 151-200
0 5 10 15 20 25 30 35
< 150
151-200 Cholesterol (mg/dl)
201-250 > 251
Fig 4 CD39 expression on platelets The analysis was done by flow cytometry (see Experimental procedures) Data represent the mean ± SE of 10 individuals Data were analyzed statistically
by Duncan’s multiple range test *Significantly different from the others (P < 0.05).
Trang 5Data from nucleotide hydrolysis and NTPDase1
(CD39) expression in platelet membranes indicated that
the mechanisms involved in ATP and ADP hydrolysis
vary with the cholesterol level In fact, up to
250 mgÆdL)1, there was no increase in CD39 expression
in platelets, but patients with cholesterol higher than
251 mgÆdL)1 exhibited a significant increase in the
expression of this complement This may indicate that
cholesterol up to 250 mgÆdL)1 causes an increase in
ATP and ADP hydrolysis by changing the plasma
mem-brane properties of platelets
In a previous study, our laboratory demonstrated
that diabetic, hypertensive and diabetic⁄ hypertensive
patients presented elevated nucleotide hydrolysis by
platelets [13,15] Taken together, we can suppose that
these associated pathologic conditions may change
the rate of platelet nucleotide hydrolysis Platelets
comprise one of the components of the thrombus
microenvironment and of the process of
thrombo-regulation It is known that the rate of platelet
nuc-leotide hydrolysis is lower in platelets than in
endothelium However, considering the function and
the mobility of platelets, we can understand their
active role in this process and the importance of
such hydrolysis In line with this, literature data
indi-cate that adenine nucleotides and adenosine are
important modulators of atherosclerosis [18], and
that upregulation of CD39⁄ NTPDase1 on platelets
has beneficial effects on endothelial cell activation;
this may be observed in vascular inflammation [19]
It is of importance that Papanikolaou et al [24]
showed that the reduction of cholesterol levels by
drugs results in strong inhibition of the enzymatic
and antiplatelet activities of NTPDase The results
presented here are in agreement with this, and we
have shown that circulating cholesterol positively
modulates platelet NTPDase1 activity Papanikolaou
et al [24] suggested that cholesterol may affect the
ability of this enzyme to undergo conformational
changes required for nucleotide hydrolysis Probably,
it enhances the interaction between the enzyme and
its substrates
In the literature, there are some studies showing that the levels of cholesterol or its oxidation status can affect ATPase activities [25,26] However, a direct role
of plasma cholesterol levels on platelet ecto-CD39 has never been established Lijnen et al [27] suggested that cholesterol lowering in hypercholesterolemic patients may result in a significant decrease in erythrocyte and platelet membrane cholesterol content Perhaps this occurred in our study When the cholesterol level is increased, the platelet membrane cholesterol content is enhanced, increasing the conformational stability of the transmembrane NTPDase1 protein, promoting activation
Taking together the importance of the physiologic (lipid bilayer component) or pathologic (factor related
to atherogenesis) functions of cholesterol, as well as the importance of the platelets and NTPDase, we con-cluded that cholesterol levels can modulate platelet NTPDase activity in vivo
In conclusion, our study demonstrated that the hydrolysis of adenine nucleotides is modified in plate-lets from hypercholesterolemic patients, and we suggest that this may play a beneficial role by preventing thrombus formation However, the modulation of nuc-leotide hydrolysis is possibly not sufficient to inhibit thrombus formation Indeed, clinical evidence indicates with clarity that the hypercholesterolemia is a risk fac-tor for future fatal events (unstable angina and myo-cardial infarction)
Experimental procedures
Chemicals Nucleotides, sodium azide, Hepes and Trizma base were pur-chased from Sigma (St Louis, MO, USA) Antibodies for flow cytometry analysis [R-phycoerythrin-conjugated mouse monoclonal antibody against human CD39, and fluorescein isothiocyanate-conjugated mouse monoclonal antibody against human CD61] were purchased from Serotec Ltd (Kidlington, Oxford, UK) and BD PharMingen (San Jose,
CA, USA), respectively All other reagents used in the experi-ments were of analytical grade and of the highest purity
Patients The sample consisted of patients with different cholesterol levels and ages ranging from 40 to 70 years, from LABI-MED (Santa Maria, RS, Brazil) We chose nonsmoking patients not undergoing hypolipemic or anti-inflammatory treatment, with glucose levels ranging from 70 to
95 mgÆdL)1 The sample was divided into four groups cons-siting of 50% female and 50% male, as follows: group 1,
Table 4 Correlation between ATP and ADP hydrolysis and
inflam-matory markers (OxLDL, hsCRP and OxLDL autoantibodies, and
triglycerides.
OxLDL autoantibodies Triglycerides
P < 0.01 P < 0.01 P < 0.01 P < 0.001
P < 0.001 P < 0.001 P < 0.001 P < 0.001
Trang 6cholesterol levels < 150 mgÆdL)1(n¼ 40); group 2,
choles-terol levels ranging from 151 to 200 mgÆdL)1 (n¼ 40);
group 3, cholesterol levels ranging from 201 to 250 mgÆdL)1
(n¼ 40); and group 4, cholesterol levels > 251 mgÆdL)1
(n¼ 40) These variations in the cholesterol values (50–
50 mg) were selected to correspond approximately to the
actual clinical interval criterion used to evaluate risk
factors The separation of the subjects into the groups was
based on clinical considerations We consider that a
cholesterol level below 150 mgÆdL)1 is low, and a level
between 151 and 200 mgÆdL)1 is clinically acceptable, but
close to the limit of 200 mgÆdL)1 A level between 201 and
250 mgÆdL)1is just above the limit, and a level higher than
251 mgÆdL)1is well above the safe limit for risk of
cardio-vascular disease All subjects gave written informed consent
to participate in the study The protocol was approved by
the Human Ethics Committee of the Health Science Center,
Federal University of Santa Maria (Protocol number:
015⁄ 2004) Eight milliliters of blood was obtained from
each participant and used for biochemical and hematologic
determinations and platelet-rich plasma (PRP) preparation
Biochemical determinations
Serum total cholesterol and triglyceride concentrations were
measured using standard enzymatic methods with the use of
Ortho-Clinical Diagnostics reagents, and a fully automated
analyzer (Vitros 950, dry chemistry; Johnson & Johnson,
Rochester, NY, USA) High-density lipoprotein cholesterol
was measured in the supernatant plasma after precipitation
of apolipoprotein B-containing lipoproteins with dextran
sulfate and magnesium chloride according to Bachorik &
Albers [28] Low-density lipoprotein cholesterol was
estima-ted with the Friedewald equation [29] hsCRP was measured
by immunoluminometry (IMMULITE 2000; Diagnostic
Products Corporation, Los Angeles, CA, USA) OxLDL was
determined by a capture ELISA according to the
manufac-turer’s instructions (Mercodia AB, Uppsala, Sweden), as
described by Holvoet et al [30] OxLDL autoantibodies were
determined using ELISA as described by Wu & Lefvert [31]
PRP preparation
PRP was prepared from human donors by the methods of
Pilla et al [21] and Lunkes et al [13] Briefly, blood was
collected into 0.129 m citrate and centrifuged at 160 g for
10 min The PRP was centrifuged at 1400 g for 15 min
and washed twice with 3.5 mm Hepes isosmolar buffer
containing 142 mm NaCl, 2.5 mm KCl, and 5.5 mm
glu-cose The washed platelets were resuspended in Hepes
buf-fer, and protein was adjusted to 0.3–0.5 mgÆmL)1, where
6–10 lg of protein was used per tube to ensure linearity
in the enzyme assay NTPDase is an ectoenzyme, and
thus platelet viability and integrity were confirmed by the
measurement of lactate dehydrogenase activity using the
enzymatic Vitros 950 (Ortho-Clinical Diagnostics; Johnson
& Johnson)
NTPDase activity Twenty microliters of the PRP preparation (10–15 lg protein) was added to the reaction mixture of NTPDase and preincubated for 10 min at 37C in a final volume
of 200 lL NTPDase activity was determined by the method of Pilla et al [21], in a reaction medium contain-ing 5.0 mm CaCl2, 100 mm NaCl, 4.0 mm KCl, 6 mm glucose, and 50 mm Tris⁄ HCl buffer (pH 7.4) The reac-tion was started by the addireac-tion of ATP or ADP as sub-strate at a final concentration of 1.0 mm The reaction was stopped by the addition of 200 lL of 10% trichloro-acetic acid to provide a final concentration of 5% The inorganic phosphate (Pi) released by ATP and ADP hydrolysis was measured by the method of Chan et al [32], using KH2PO4 as a standard Controls were pre-pared to correct for nonenzymatic hydrolysis by adding PRP after trichloroacetic acid addition All samples were run in triplicate Enzyme activities are reported as nmol
Pi releasedÆmin)1Æmg)1 protein
Flow cytometry analysis Peripheral blood cells were incubated with anti-CD39 and anti-CD61 (20 lL per 106 cells) for 25 min, lysed with fluorescence activated cell sorter (FACS) reagent, and incu-bated again for 15 min in the dark Cells were washed twice
in NaCl⁄ Pibuffer (pH 7.4) containing 0.02% (w⁄ v) sodium azide and 0.2% (w⁄ v) BSA The cells were then
resuspend-ed in NaCl⁄ Pi buffer (pH 7.4) and immediately analyzed with a FACScalibur flow cytometer using cellquest software (Becton Dickinson, San Jose, CA, USA), without fixation
Hematologic determinations Quantitative determinations of platelets obtained by veni-puncture were performed using a Pentra 120 analyzer (ABX, Montpellier, France) Platelet aggregation was per-formed by the technique of Biggs [33], consisting of the
in vitro macroscopic visualization of aggregates at intervals
of 15–50 s by the addition of ADP to PRP
Determination of lipid peroxidation Lipid peroxidation was estimated by the measurement of thiobarbituric acid reactive substances in serum samples by modifications of the method of Jentzsch et al [34] Briefly, 0.2 mL of serum was added to the reaction mixture con-taining 1 mL of 1% orthophosphoric acid, and 0.25 mL of
an alkaline solution of thiobarbituric acid (final volume
Trang 72.0 mL), and this was followed by 45 min of heating at
95C After cooling, samples and standards of
malondial-dehyde were read at 532 nm against the blank of the
stand-ard curve The results were expressed as nmol
malondialdehydeÆmL)1
Protein determination
Protein was determined by the Coomassie blue method
using BSA as standard [35]
Statistical analysis
Data were analyzed statistically by one-way anova
fol-lowed by the Duncan test Differences between groups were
considered to be significant when P < 0.05 Linear
correla-tion between variables was also carried out
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