Probucol Reduces Myeloperoxidase Expression in Peripheral Blood Monocytes from Patients with Acute Coronary Syndrome
X Xie, G Huang, S Zhao
atherosclerosis, inflammation, myeloperoxidase, probucol
X Xie, G Huang, S Zhao. Probucol Reduces Myeloperoxidase Expression in Peripheral Blood Monocytes from Patients with Acute Coronary Syndrome. The Internet Journal of Cardiology. 2009 Volume 8 Number 1.
Increasing evidences have supported the notion that inflammation is involved in the atherogenesis and pathogenesis of acute coronary symdrome (ACS) . Multiple lines of evidence have suggested that myeloperoxidase (MPO) might play an important role in atherosclerosis . Higher plasma levels of MPO were correlated with increased incidence of coronary artery disease (CAD)  and cardiovascular events. Patients who were deficient in MPO were less susceptible to cardiovascular diseases. A functional MPO promoter polymorphism −463G/A, which altered MPO expression levels [5-6], was associated with increased incidence of CAD and severity of atherosclerosis [7-9]. Treatment with antioxidants may affect the expression of MPO in peripheral blood monocytes (PBMs) .
Nitric oxide (NO) plays essential bioregulatory roles in a wide range of processes . Under pathological conditions, such as in inflammation and vascular diseases, rates of NO consumption became excessive and the response to endothelium-derived relaxing factor was impaired [12-13]. MPO is a major scavenger and a marker of oxidative stress. Superoxide, NO and MPO are seemed to be coupled through complex and inter-dependent pathway. The biological consequence of NO-peroxidase interactions may have broad implication for regulation of inflammatory and cardiovascular events in vivo.
Probucol is a known lipophilic antioxidant with modest lipid-lowering properties. Among the antioxidants relevant to cardiovascular disease, probucol is of particular interest , as it inhibits atherosclerosis in hypercholesterolemic rabbits[17,18], non-human primates, and humans, probucol also reverses estabolished plaques in rabbits and xanthomas in humans and it effectively inhibits intimal thickening and restenosis [23,24], preserves endothelial function，it inhibits atherosclerosis in some animals and can decrease the complications of cardiovascular disease in patients with hypercholesterolemia. In the Fukuoka Atherosclerosis Trial (FAST), probucol lowered cholesterol and stopped progression of atherosclerotic plaques in carotid arteries . Our previous study had shown that probucol up-regulated paraoxonase 1 expression in hypercholesterolemic rabbits, which might be contributed to its antiatherogenic effect . Paul et al  showed that probucol protected against HOCL-induced endothelial dysfunction. However, the exact mechanisms underlying its vascular protective activity were remained unclear. Therefore we decided to test the effect of probucol on the expression of MPO in PBMs from patients with ACS and SCHD , and to examine whether the benefit activity of probucol was related to another inflammatory factor-MPO.
Materials and methods
We recruited 35 patients with acute coronary syndrome (ACS group) including acute myocardial infarction and unstable angina. 20 patients with stable coronary heart disease (SCHD group) of matched age were treated as control. Those patients with unstable angina had experienced ischemic chest pain at rest accompanied with transient ST-T segment depression and/or T-wave inversion within the proceeding 24 h. The diagnosis of acute myocardial infarction was based on a history of ischemic chest pain > 30 min, characteristic ECG changes, and increasing of creatine kinase activities at least twice the upper normal level within 24 h after the onset of pain. Stable CHD was defined as clearly established CHD with no history of recent hospital admission (within the previous 3 months) for treatment of a coronary condition. All patients underwent coronary angiography , and Gensini scores were used to evaluate coronary severity. Exclusion criteria included body temperature > 38°C, inflammatory diseases (e.g., infections, malignancies, autoimmune diseases), impaired liver function, renal failure, serum total cholesterol concentration > 7.0 mmol/L, and recent major surgery. PBMs were collected from all participants, and the monocytes from patients with ACS were collected within 12 h after the onset of pain. Serum levels CRP as well as lipid profiles, liver and kidney function, cardiac enzyme (CK and CK-MB) were obtained in the fasting state the first day. All the venous blood samples were drawn into pyrogen-free blood collection tubes without additive. The serum was collected after centrifugation at 3500 rpm for 3 min and then stored at −70°C until analyzed. Serum CRP levels were determined by turbidimetric immunoassay (CRP kit, Beckman Coulter), and the examinations were done in Array 360 Analyzer (Beckman Coulter). Total cholesterol (TC) and triglyceride (TG) concentrations were analyzed using a Hitachi 747 autoanalyzer (Boehringer Mannheim, Naka, Japan). Serum glucose was measured using the glucose oxidase method. MPO levels in plasma were measured by an ELISA test kit (Corgenix,Inc,USA). The study was approved by the local Institutional Review Board.
PBMs isolated and culture
Peripheral venous blood from the patients with ACS (15 mL) or with stable CHD (5 mL) was layered on an equal volume of Ficoll Hypaque (Shanghai Biotech) and was centrifuged at 1500g for 20 min at 25 °C. PBMs were harvested from the Ficoll-plasma interface and were washed twice with cold Hanks’balanced salt solution (Sigma) and then once with serum-free RPMI 1640 (Gibco Life Technologies). The final pellet was suspended in serum-free RPMI 1640, and the cells were plated in 75-cm2 flasks at a density 2 x 107 cells/dish. Cell viability, as assessed by trypan blue exclusion, was routinely >95%. Nonadhering cells were removed after 6 h of incubation. Adhering monocytes were cultured for 24 h with RPMI 1640 supplemented with 2mmol/L N-acetyl-L-alanyl-L-glutamine, 100 kilounits/L penicillin, 100 mg/L streptomycin, 20 g/L sodium pyruvate, 20 mmol/L HEPES (Gibco Life Technologies), and 100 mL/L heat-inactivated fetal calf serum (Gibco Life Technologies) at 37°C in 5% CO2. Cell culture supernatants were collected and stored at –70°C for MPO assays. Adhesive monocytes were used to extract total protein for Western blot. For in vitro studies, PBMs from patients with ACS were isolated as described above. After removal of the nonadhering cells, monocytes were exposed to probucol(0，5，10 µmol/L) dissolved in dimethyl sulfoxide (Sigma) for 24 h, and cells were collected for MPO assays, and supernatants were collected for detection of MPO levels , NO concentration , as well as iNOS activity.
Detection of MPO expression in cultured PBMs by Western blot analysis
To investigate the expression of MPO in human PBMS, immunoblot analysis were performed. Cells (2×107) were lysed with Triton lysis buffer (50 mM Tris-HCL, pH 8.0 containing 150 mM NaCL, 1% Ttriton X-100, 0.02% soldium azid, 10 mM EDTA ,10 µg/ml aprotinin, and 1µg/ml aminoethylbenzenesulfonyl fluoride). Protein concentrations were determined by Bradford assay. One hundred micrograms protein from cell lycates were loaded onto a 7.5% SDS-PAGE gel. After electronphoresis, the SDS-PAGE separated proteins were transferred to a nitrocellulose membrane (Amersham Pharmacia Biotech).The membrane was blocked with 2.5% nonfat milk in PBS and incubated with rabbit antibody against human MPO(1:200 dilution) in PBS for 2 h. Then the membrane was incubated with goat anti-rabbit IgG conjugated with horseradish peroxidase (Santa Cruz,Biotechnology, CA, USA) at 1:1000 in PBS for 1h. Blots were processed using an ECL kit (Santa Cruz) and exposed to X-ray film.
MPO levels in supernatants were measured by an ELISA test kit (Corgenix,Inc,USA).Concentration of nitrites in cell supernatants was measured as an indicator of NO production according to the method of Gilliam et al. Briefly, the total concentration of nitrites was determined colorimetrically with the Griess reagent. The absorbance was measured at 546 nm (Spectra Rainbow,SLT,Austria). Sodium nitrite was used as a standard. NOS activity was evaluated by measuring the rate of conversion of L-[U-14C]arginine to citrulline, according to the method of Salter et al. The activity was expressed as nmol/min/ml of supernatant.
Student’s t test or analysis of variance (ANOVA) was used to determine statistical significance of probucol effects using SPSS 15.0 (SPSS software). All values are expressed as means±SEM. A probability less than 0.05 was considered significant. The association of measurements with other biochemical variables was assessed by the Spearman rank correlation test.
Baseline characteristics of patients with ACS and SCHD
The baseline characteristics of the patients are summarized in Table 1. Sex distribution, age, body mass index, smoking, fasting blood glucose, creatinine, TG, TC 、Gensini scores 、LVEF and blood pressure were not significantly different among the groups; while white blood cells 、PBMC-MPO expression、serum-MPO and CRP were significantly higher in ACS group as compared with SCHD group. There was significant negative correlation between PBMC-MPO expression and LVEF for all patients(r =-0.702, P<0.001), However it was not correlated with plasma CRP concentration (r=0.488, p >0.05)、 serum-MPO(r=0.287, p >0.05), and no statistically significant correlation was found between PBMC-MPO expression and Gensini scores(r =0.160, P>0.1) .
As compared with SCHD group, * p<0.05.
Values are presented as mean ± SEM. or
BMI: Body Mass Index; WBC: white blood cell; TG: triglyceride; TC: total cholesterol;
CRP: C Reactive Protein.
A: Detection of MPO expression in cultured PBMs from patients with ACS and SCHD by Western blot analysis. Lane 1: probucol pretreatment (0µmol/L); Lane 2: probucol 5µmol/L; Lane3: probucol (10µmol/L); Lane 4: SCHD group.
*:indicates a significant difference (
** indicates a significant difference (
MPO expression in PBMs is expressed as absorbance ratio of MPO product to β-actin product; concentration of nitrites in cell supernatants was measured as an indicator of NO production according to the method of Gilliam et al. r = -0.548, p<0.001.
Local and systemic inflammatory responses are commonly involved in the progress of ACS. PBMs are activated after ACS, and these inflammatory cells are important in blood prone to thrombosis (vulnerable blood).MPO which is largely abundant expressed in neutrophils and monocytes catalyzes the formation of many reactive oxidant species [30-31]. MPO promotes atherogenesis via a range of mechanisms. One of the major mechanisms is that MPO can utilizes the nitric oxide (NO) as substrate to generate NO-derived oxidants, which leads to endothelial dysfunction, accumulation of foam cells in the arterial wall and the promotion of plaque rupture . Evidence suggests that MPO-generated reactive nitrogen species can convert native low density lipoprotein (LDL) into an atherogenic form, promote the peroxidation of lipids . It is also appeared that MPO can selectively modify apolipoprotein A-I, impair ABCA-1 dependent cholesterol efflux . Moreover, studies suggest MPO plays a potential role in plaque destabilization and rupture due to activation of matrix metalloproteases (MMPs). In addition to , Sugiyama et al. demonstrated that HOCl could augment tissue factor (TF) expression in human EC and provoke human EC death and detachment through both apoptotic and oncotic cell death pathways. Their results suggested that MPO-derived HOCl might participate in ACS by promoting superficial erosion and increasing thrombegenicity. In our study, PBMC- MPO expression and MPO levels in supernatants were significantly higher with ACS as compared those with SCHD, while Gensini scores were not significantly different among these two groups, which is consistent with the results of other authors, indicate that MPO takes part in the acute complication phases of the atherosclerotic process; and there was significant negative correlation between PBMC-MPO expression and LVEF for all patients(r =-0.702, P<0.001), indicate that MPO may impaired heart function. As a result, MPO and its downstream inflammatory pathways may become attractive targets for both prognostication and therapeutic intervention in the atherosclerotic cardiovascular disease.
Probucol was originally used as hypolipidemic drug to prevent atherosclerosis, due to its inhibitory effect on the oxidation of LDL. Recently, several studies suggested that probucol might also have anti-inflammatory actions to explain its anti-atherosclerotic activity by reducing vascular cell adhesion molecule-1 and monocyte chemoattractant protein-1, up-regulating paraoxonase-1 expression and inducing heme oxygenase-1 paraoxonase-1 expression . In the present study, we demonstrat, for the first time, probucol can down-regulate MPO expression in PBMs and reduced the MPO levels in supernatants with ACS. Though there were some discrepancies between the MPO expression in PBMs and the secretion into the supernatant among ACS patients, these discrepancies may be due to the concentrations of probucol, at 10µmol/L or higher, it inhibits both expression and secretion. Accordingly, probucol significantly increase NO levels in PBMs from patients with ACS without affecting iNOS. The MPO expression in PBMs from patients with ACS is negatively correlated with NO levels in supernatants, indicating that probucol reduce NO removal by MPO, and it can not produce more NO from monocytes ( because without affecting iNOS). All of these results suggest that probucol maybe play anti-atherosclerosis effects by reducing MPO expression in PBMs. It is not known how probucol could modify the expression of MPO in monocytes. It has been suggested that some inflammatory genes transcription and expression are regulated through an antioxidant-sensitive mechanism in HUVEC. By actively inhibiting NF-kappaB mobilization , antioxidants like NAC or PDTC can reduce the expression of some inflammatory factors, such as VCAM-1,TNF-kappaB,IL-1 or MPO[39,40,41], probucol, a potent antioxidant, may also suppress inflammatory factors through the same mechanism. Recent evidence indicates that agonist for the peroxisome proliferators-activated receptor (PPAR) regulate MPO gene expression via the aforementioned ALuRRE[42,43], while our laboratory previous study reported that probucol suppressed NF-κΒ-p65 expression induced by Ang Ⅱ,along with increases in the expression of PPAR. Therefore, the mechanism which probucol reduced the MPO expression and secretion is probably related to NF-kappaB or PPAR .
ACS might relate to monocytes activation and MPO increasing. In our present study , MPO expression in PBMs with ACS were markedly increased, probucol can decreased MPO expression and secretion in PBMs in patients with ACS, it may also decrease MPO levels in serum . therefore, treatment with probucol may be a new way in ACS .
In conclusion, we have been able to show a novel target site for the protective action of probucol, in which this drug can reduce MPO expression in PBMs with ACS. Further clinical studies will be required to substantiate whether probucol may influence on plasma MPO levels with ACS patients and whether treatment with probucol can improve heart function.