Abstract
Background and Aims
Plasma concentrations of anti‐inflammatory cytokine interleukin 10 (IL10) have been shown to be decreased in patients with unstable angina (UA) suggesting that reduced concentrations of IL10 may favour plaque instability and the development of acute coronary syndromes. Diltiazem has been shown to exert beneficial effects in patients with acute coronary syndrome. However, the potential influence of diltiazem on the anti‐inflammatory cytokine IL10 in patients with UA has not been investigated. This study was designed to find out the effects of diltiazem on IL10 in UA patients.
Methods and Results
Thirty patients with UA were divided into two groups: group R and group D (n = 15). Group R was given routine pharmacotherapy for UA, and group D was given routine pharmacotherapy plus diltiazem. Plasma concentrations of IL10 in these groups were measured before the start of the treatment and 28 days after treatment. Plasma concentrations of IL10 in 15 normal subjects (group N) were also measured. Patients with UA had decreased concentrations of IL10 compared with normal group. Four weeks after treatment, plasma concentrations of IL10 significantly increased in group D compared with that before treatment, but the increase in IL10 values in group R was not significant.
Conclusions
These findings showed that concentrations of anti‐inflammatory IL10 are considerably decreased in UA patients and diltiazem treatment leads to a significant increase in IL10 concentrations.
Keywords: diltiazem, interleukin 10, unstable angina
Clinicopathological studies over many years have shown that coronary plaque rupture containing a lipid rich core with subsequent thrombus formation is the most important mechanism by which atherosclerosis leads to the acute ischaemic syndromes of unstable angina (UA), acute myocardial infarction (MI), and sudden cardiac death.1 Growing evidences have shown the importance of inflammation in the process of atherogenesis as well as in its progression and the determination of plaque vulnerability.2,3,4 Even the earliest manifestation of atherosclerosis, fatty streak, is an inflammatory lesion that consists of macrophages and T lymphocytes.5 Studies further suggested the role of T cells in atherosclerosis as well as UA. These cells secrete different kinds of cytokines, both proinflammatory as well as anti‐inflammatory, that have been seen to be associated with atherosclerosis and its progression to the clinical syndromes.
Increased circulating concentrations of proinflammatory cytokines, including interleukin (IL) 1ß, IL6, IL12, interferon gamma, and tumour necrosis factor α have been found in patients with acute coronary syndromes (ACS). In contrast, cytokines such as IL10 and IL4 have been implicated as anti‐inflammatory cytokines and were found in decreased concentrations in patients with UA than normal subjects as well as those having stable angina. Serum concentrations of the potent anti‐inflammatory cytokine IL10 have recently been shown to be decreased in patients with ACS6 and increased serum concentrations of IL10 were associated with a significantly improved outcome for patients with ACS,7 thus suggesting that reduced values of IL10 may favour plaque instability and the development of ACS.
Emerging data have shown that diltiazem, a non‐dihydropyridine calcium channel blocking agent, plays a beneficial part in patients with UA. Patients with UA are generally considered to be at risk of recurrent and possibly irreversible ischaemia, resulting in progression to MI or sudden cardiac death. Therefore, the objectives of treatment should not be only pain relief but also prevention of recurrent ischaemia or MI. Diltiazem has been used extensively as a vasodilator in the management of UA. Data from the diltiazem reinfarction study in patients with non‐Q wave MI8 showed reduced rates of reinfarction and severe angina during 14 days of treatment with diltiazem. Improved prognosis for coronary artery disease (CAD) after diltiazem treatment has been attributed to its vasodilatory property. But, it is now supposed that diltiazem might exert some action other than vasodilatation that is responsible for the remission of UA and improved prognosis.9 Recent evidences have shown that diltiazem has anti‐inflammatory actions. Its ability to downregulate T, B, and NK cell proliferation and function was widely shown several years ago. Moreover, in a recent publication it was shown that diltiazem is able to modify dendritic cell (DC) functions through its pronounced downregulation of antigen presenting molecules, costimulatory molecules, and inhibition of IL12 production.10 It is reported that function of DC is increased in patients with UA and the activated function of DC is an important mediator in the inflammatory process as they are responsible for T cell activation, and this leads to plaque instability and vulnerability towards rupture.11 DCs express costimulatory molecules including CD80 (B7‐1), and CD86 (B7‐2), which are upregulated during DC activation, and this explains their unique ability to activate naive T cells. Costimulatory signal mediated by the interaction of CD86 (B7‐2) with CD28 is important for the activation of T cells stimulated with antigen on antigen presenting cells,12 and it has been shown that Ag presentation in the absence of CD86/CD28 costimulation have a direct negative impact on T cell activation.
These findings led us to hypothesise that diltiazem might also exert an anti‐inflammatory role in UA. However, the potential influence of diltiazem on the anti‐inflammatory cytokine IL10 in patients with UA was not investigated. Therefore, we designed this study to investigate the effect of diltiazem on IL10 in UA patients.
Methods
Patient selection
Thirty patients admitted with a diagnosis of primary UA in our department (department of cardiology, Tongji Hospital) were included in this study. These patients had chest pain at rest and were admitted within 24 hours of the last episode of symptom onset, thus belonged to class III of Braunwald classification for severity of UA.13 All patients had diagnostic ST segment displacement >0.01 mV, and without any increase in biomarkers for MI (CK, CK‐MB, MYO, and TnI). They were divided into two groups (each group with 15 patients). Fifteen were given routine pharmacotherapy without diltiazem and grouped as R, and 15 were given routine pharmacotherapy plus diltiazem, and grouped as D. Fifteen healthy subjects who visited the same hospital for routine physical check up were also enrolled as normal control group and grouped as N. These subjects did not give any history of chest pain or symptoms suggestive of CAD. They were also screened non‐invasively through clinical assessment, ECG, and exercise testing to rule out the presence of CAD. These subjects also showed no evidence of ongoing systemic or cardiac inflammatory diseases. All patients were enrolled in this study after informed consent had been obtained from them. Transthoracic echocardiography was performed in all patients to exclude patients with an impaired left ventricular ejection fraction (<50%) as well as left heart chamber dilatation. The patients included in our study were not taking any kind of calcium channel blocking agents. Patients in group D were enrolled for the study only if they did not have any of the following:
(1) Acute myocardial infarction on admission (cardiac enzymes activities increasing from normal value at admission and ST segment elevation in ECG). (2) Heart rate <50 bpm. (3) Systolic blood pressure <90 mm Hg. (4) Any degree of AV block. (5) Severe heart failure (NYHA class III‐IV). (6) Sick sinus syndrome. (7) Atrial fibrillation or atrial flutter. (8) Intraventricular conduction disturbances (QRS >100 ms). (9) Known intolerance of calcium channel blockers.
Treatment
Group R was given routine pharmacotherapy for UA without diltiazem. Group D was given routine pharmacotherapy with diltiazem. Routine pharmacotherapy includes antiplatelet (aspirin, ticlopidine, or clopidogrel), anticoagulant (LMWH), β blocker, and nitrates. Diltiazem (Herbesser, Tianjin, China) was given by oral dose of 30–60 mg thrice daily. Oral dose of diltiazem was kept for 28 days. A volume of 500 ml of NS was rapidly given when symptomatic hypotension or heart rate <50 bpm was experienced by patients. In the absence of improvement or the presence of more severe bradyarrhythmia such as second degree or third degree AV block, patients were excluded from the group D. Only the patients with hyperlipidaemia were given statins (three in group R and two in group D) because the influence of statins on immune cells and inflammatory cytokines production has been shown in some studies.
Peripheral blood was collected before treatment and four weeks after treatment from group R and group D. Blood from group N was collected just once.
Laboratory analysis
Blood samples were obtained from UA patients (group R and group D) immediately after admission and four weeks after treatment. Samples were collected into collection tubes containing EDTA and the plasma thus obtained, after centrifugation at 3000 rpm, were stored at −80°C for the subsequent analysis of the IL10. Blood samples from group N was taken once in the morning after an overnight fast and then collected and stored in a similar way mentioned above for the analysis of the cytokine. We measured the plasma concentrations of IL10 by sandwich enzyme linked immunosorbent assay (ELISA) kits (Biosource International, CA, USA). The lower limit of detection for IL10 was <1 pg/ml. The inter‐assay and intra‐assay variation coefficients were ⩽5.5% and ⩽4.8% respectively. All the laboratory procedures were conducted according to the manufacturer's instructions by an experienced technician who was unaware of this study.
Statistical methods
Values are presented as mean (SD). SPSS 13.0 software was used for statistical analysis. A t test was performed for comparison between the two groups. A p<0.05 was considered to be significant.
Results
Thirty consecutive patients with diagnosis of UA who meet the criteria for enrolment, and 15 normal subjects who visited our hospital for routine physical check up were selected for this study. Eighty per cent in the UA group and 74% in normal control group were male. Table 1 lists the clinical and demographic data of each group. The prevalence of hypertension and current smoking was significantly higher in the UA patients (group R and group D) compared with the normal control (group N). During the study, three patients in group D and two in group R experienced hypotension (SBP <100 mm Hg) or bradycardia (HR <50 bpm), or both, and they recovered after administration of intravenous fluid. Other side effects such as second degree or third degree AV block were not noted in the group R. However, one patient was excluded from group D because of second degree AV block, accompanied by dizziness.
Table 1 Clinical and demographic data of the study groups.
| Group N | Group R | Group D | |
|---|---|---|---|
| Mean age (years) | 60 (3) | 61 (5) | 61 (6) |
| Men (n) | 11 (74%) | 12 (80%) | 12 (80%) |
| HTN (n) | 6 (40%) | 10 (67%)* | 11 (74%)* |
| DM (n) | 4 (27%) | 5 (34%) | 5 (34%) |
| Hyperlipidaemia (n) | 3 (20%) | 3 (20%) | 2 (14%) |
| Current smoking (n) | 3 (20%) | 9 (60%)* | 10 (67%)* |
| Echocardiography | |||
| Diameter of left atrium (mm) | 31.05 (4.21) | 32.14 (4.30) | 31.23 (4.02) |
| Diameter of left ventricle (mm) | 48.25 (7.45) | 49.34 (8.06) | 48.39 (8.19) |
| Ejection fraction (%) | 60.12 (11.42) | 59.29 (12.20) | 58.34 (11.31) |
*p<0.01 v group N. Data are mean (SD) unless shown otherwise.
Comparison of IL10 among the groups
The concentrations of IL10 in unstable angina group (group R and group D) before treatment and group N are given in table 2 and figure 1. The concentrations of IL10 were significantly lower in the UA groups before treatment compared with the group N. The difference in the cytokines concentrations between group R and group D was obviously not significant.
Table 2 Comparison of IL10 in group N and patient groups (groups R and D) before treatment and after treatment.
| Group R (BT) | Group D (BT) | Group N | Group R (AT) | Group D (AT) | |
|---|---|---|---|---|---|
| IL10 | 6.75 (2.67)** | 6.82 (2.68)** | 16.47 (3.94) | 8.21 (2.69) | 12.95 (3.73)* (pg/ml) |
BT, before treatment; AT, after treatment. **p<0.01 v group N. *p<0.05 v group D before treatment.
Figure 1 Comparison of IL10 in group R and group D before treatment (BT) with group N. **p<0.01 v R (B) and D (B).
After treatment, the concentration of IL10 was significantly higher in the group D compared with that before treatment. However, no such change in IL10 was seen in the group R after treatment (p>0.05). The concentrations of IL10 in these groups after treatment and comparison with that before treatment are given in table 2 and figure 2.
Figure 2 Comparison of IL10 in group D and group R after treatment (AT) with that before treatment (BT). Concentration of IL10 in group R (AT) was not significant (NS) compared with that before treatment R (AT). *p<0.05 v D (B).
Discussion
Proinflammatory cytokines have the potential to induce excessive extracellular matrix degradation and cell death thus can play important part in plaque instability and vulnerability toward rupture.14 They may also be responsible for the activation of other cells such as, neutrophils, macrophages, or mast cells. However, the inflammatory response is known to be balanced by anti‐inflammatory cytokines. One such cytokine IL10, has been seen to downregulate the proinflammatory roles of macrophages15 and inhibit interferon gamma producing function of Th1 cells.16 It is well established that IL10 is secreted by activated macrophages and T lymphocytes. The anti‐inflammatory role of IL10 was studied both in experimental models and human subjects. Studies have shown that serum IL10 concentrations are significantly lower in UA patients and decreased serum concentration of IL10 is associated with ACS,6,7,17 which is also supported by the findings of our study. Our study showed that concentration of IL10 is significantly lower in UA patients than in normal controls. IL10 inhibits many cellular processes that could play an important part in plaque progression, rupture, or thrombosis, including macrophage function, production of proinflammatory cytokines, NF‐κB activation, MMP production, and cell death.
Diltiazem is a calcium antagonist widely used clinically because of its properties of vasodilatation and modest negative inotropic effect and nodal inhibition. In a double blind randomised trial in UA it was shown that intravenous diltiazem reduced ischaemic events better than intravenous nitroglycerin.18 Several others trials conducted in patients with non‐transmural MI and UA showed that long term oral diltiazem significantly improves the prognosis and event free survival.8,19,20 Theroux et al in DATA trial showed that intravenous diltiazem for 48 hours along with fibrinolytic agent then followed by oral administration of diltiazem for four weeks significantly reduced the rate of death, reinfarction, or recurrent ischaemia.21 It has been shown that patients receiving diltiazem had significant reduction in enzyme release as well as a reduced prevalence of “reperfusion” arrhythmias.22
Many studies have shown anti‐inflammatory effects of diltiazem. Because of its immunomodulatory and immunosuppressive effects, diltiazem has also been used in transplantation. Diltiazem treatment to the patients undergoing cardiopulmonary bypass significantly reduced IL6 while upregulated IL10 concentrations at the end of cardiopulmonary bypass.23 It has been shown that calcium channel blockers modulate the production of some proinflammatory and anti‐inflammatory mediators in endotoxaemia.24 In that study it was shown that lipopolysaccharide induced IL10 plasma concentrations were significantly increased, and circulating TNFα concentrations were significantly suppressed in animals pretreated intraperitoneally with verapamil or diltiazem. More recent studies have reported an immunomodulatory effect of diltiazem on cytokine production by different cell types, including human monocytes and DCs25,26 Diltiazem has been shown to affect human DC maturation in vitro and downregulate IL12 production by human DCs.27 Although numerous previous studies have shown anti‐inflammatory effects of diltiazem, the anti‐inflammatory effect of diltiazem in patients with UA was not studied. Our data showed that serum IL10 concentration was significantly low in UA patients and diltiazem treatment considerably increased the concentration of anti‐inflammatory cytokine IL10. Furthermore, we found that increase in the concentration of IL10 was not significant in those patients receiving routine therapy without diltiazem. However, the data did not show that diltiazem directly influences the production of IL10. Exact mechanism how diltiazem increases IL10 concentration in UA should be studied further. The patient groups in our study also received β blocking agent. Notably, β blockers have been shown to lower the plasma concentrations of IL10. Ohtsuka et al have shown that β blockers lower circulating concentrations of TNFα, and that the reduction of TNFα concentrations is closely related to the change in the level of IL10.28 We saw that increase in the concentration of IL10 was not significant in those patients who received routine therapy without diltiazem. However, circulating IL10 was significantly increased in those who received diltiazem, suggesting that diltiazem exerts anti‐inflammatory effects in UA. The possible mechanism might be the effect of diltiazem in DC maturation and costimulatory molecules expression on DC or direct inhibitory effect on macrophages or T lymphocytes. Ability of diltiazem to downregulate T, B, and NK cell proliferation and function was widely shown several years ago.29 Studies have also found that diltiazem affects human DC maturation and downregulates CD86 costimulatory and HLA‐DR molecules. CD86/CD28 pathway is the dominant costimulatory pathway, and has a critical role in the regulation of T cell viability, cytokine production, clonal expansion, and effector function and this cellular interaction plays a part in plaque instability and vulnerability towards rupture. Studies have shown that costimulatory molecules CD86 are upregulated in patients with UA compared with normal.11 Downregulation of CD86 costimulatory molecule expression on DC thus inhibits the ability of DCs to stimulate T cell activation and reduces the secretion of proinflammatory cytokines while increases anti‐inflammatory cytokine IL10 concentrations.
The findings in this study suggested that the concentrations of IL10 is lowered in unstable CAD and diltiazem treated patients have significantly increased concentrations of anti‐inflammatory cytokine IL10 than controls and thus diltiazem might play an important part in inhibiting plaque instability and vulnerability towards rupture. However, the numbers of patients included in this study were limited and cardiovascular events were also not evaluated. Therefore, this study does not allow us to reach concrete conclusion. Future research in this field will have to focus more precisely on the effects of diltiazem on different inflammatory markers and the possible anti‐inflammatory mechanism in UA.
Abbreviations
UA - unstable angina
ACS - acute coronary syndrome
MI - myocardial infarction
IL - interleukin
TNF - tumour necrosis factor
CAD - coronary artery disease
DC - dendritic cell
Footnotes
Funding: none.
Conflicts of interest: none.
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