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- Materials and methods
The aim of the present study was to explore the relationship between tissue levels of leptin, soluble interleukin-6 receptor (sIL-6R), high-sensitive-C-reactive protein (hs-CRP) and soluble vascular cell adhesion molecule-1 (sVCAM-1) in atherosclerotic plaques, and traditional risk factors. Coronary artery specimens were obtained from 35 consecutive patients (26 men and nine women) who underwent coronary artery bypass grafting procedure. The mean tissue levels of leptin, hs-CRP and sIL-6R were significantly higher in patients with diabetes mellitus than without diabetes mellitus. When patients were classified according to the smoking status, the mean tissue levels of leptin, hs-CRP and sIL-6R were significantly higher in current smokers than both former smokers and non-smokers. In addition, the mean tissue levels of leptin and sIL-6R were significantly higher in former smokers than non-smokers. There was a positive association between leptin and hs-CRP, sIL-6R and plasma glucose in all patients. Plasma HDL levels were associated negatively with atherosclerotic tissue levels of leptin. Tissue levels of sIL-6R were associated significantly in a positive manner with leptin, hs-CRP and plasma glucose, while tissue levels of hs-CRP were associated with both leptin and sIL-6R. In conclusion, it is attractive to speculate that hs-CRP, sIL-6R and leptin could act synergistically in course of local inflammatory activity and those molecules may not be just markers of inflammation and cardiovascular risk but are also likely to play a pathogenic role in atheromatous plaque. In addition, atherosclerotic tissue levels of CRP, sIL-6R and leptin were significantly higher in current smokers and patients with diabetes.
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- Materials and methods
There is clear evidence today that the monocyte-derived macrophages, T lymphocytes and a large number of proinflammatory cytokines play a key role in all the phases of atherosclerosis [1,2]. In this context, the atherosclerotic plaque may be viewed as a chronic inflammatory nidus that is filled with immune cells that can orchestrate and effect inflammatory responses.
Leptin was thought originally to be an anti-obesity hormone, but it is also a crucial molecule for a number of diverse physiological processes, such as inflammation , immune function  and atherosclerosis . Previous data indicate that hyperleptinaemia is involved in the pathogenesis of atherosclerosis [6,7].
Interleukin (IL)-6 plays a central role in various host defence mechanisms, such as the general immune response, acute phase reactions and haematopoiesis . The biological activity of IL-6 is mediated via two membrane receptor proteins, a unique low-affinity binding receptor (IL-6R) and the high-affinity transducing β-subunit gp130. Although IL-6 may bind the IL-6 receptor (IL-6R) and elicit a biological response, it also induces the release of soluble IL-6 (sIL-6R). Recent evidence indicates that the pathophysiological effects of IL-6 may depend strongly on a soluble form of the receptor . It has been demonstrated that IL-6 and sIL-6R are associated with the processes of inflammation and myocardial injury during the acute phase of acute myocardial infarction . Furthermore, IL-6 gene transcripts are expressed in human atherosclerotic lesions [11,12].
Experimental studies in vitro have generated direct evidence that C-reactive protein (CRP) is involved in the pathogenesis of atherosclerosis [13,14]. CRP also appears to be produced locally in atherosclerotic plaques by resident macrophages and vascular smooth muscle cells and may be involved in several important steps in plaques genesis and progression . Levels of CRP mRNA and protein are increased in atherosclerotic plaque obtained from autopsy samples . In a recent report, Ishikawa et al. demonstrated that the gene for CRP was expressed in coronary plaque tissue obtained during the atherectomy procedure .
Vascular cell adhesion molecule-1 (VCAM-1) is an endothelial adhesion molecule of the Ig gene superfamily that has been considered as a biomarker of atherosclerosis . VCAM-1 is involved primarily in the adhesion of mononuclear leucocytes to the endothelium, and is induced rapidly by the proinflammatory cytokines. O’Brien et al. have observed a striking association between the degree of macrophage accumulation and expression of VCAM-1 on neovasculature and on neoendothelial cells of atherosclerotic plaques . VCAM-1 has been found to play a role in the initiation of atherosclerosis in mice .
Coronary atherectomy specimens provide a unique source of atherosclerotic plaque tissues because they make it possible to correlate tissue concentrations of inflammatory markers with the clinical status of the patient. The aim of the present study was to explore the relationship between tissue levels of leptin, sIL-6R, hs-CRP and sVCAM-1 in atherosclerotic plaques, and traditional risk factors.
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- Materials and methods
Clinical and laboratory characteristics of study participants are shown in Table 1. Hypercholesterolaemia was present in 10 (28·6%) patients, hypertension in 18 (51·4%), diabetes mellitus in 14 (40·0%), family history of CHD in 15 (42·9%) and prior myocardial infarction (MI) in 16 (45·7%). At the time of the study, the patients were being treated with aspirin (n = 35, 100%), beta-blockers (n = 22, 62·9%), angiotensin-converting enzyme inhibitors/angiotensin-II receptor blockers (n = 18, 51·4%), oral anti-diabetics (n = 14, 40·0%), nitrates (n = 13, 37·1%), calcium antagonists (n = 13, 37·1%) and statins (n = 7, 22·9%).
Table 1. Clinical and laboratory characteristics of study participants (n = 35).
|Factor||Mean ± s.d.|
|Age, years|| 58·40 ± 9·85|
|Male, %|| 74·29|
|Body mass index, kg/m2|| 23·86 ± 3·42|
|Systolic blood pressure, mmHg||135 ± 19|
|Diastolic blood pressure, mmHg|| 80 ± 10|
|Total cholesterol, mmol/l|| 4·50 ± 1·10|
|LDL cholesterol, mmol/l|| 2·83 ± 0·90|
|HDL cholesterol, mmol/l|| 1·00 ± 0·21|
|Triglycerides, mmol/l|| 1·45 ± 0·59|
|Fasting glucose, mmol/l|| 6·84 ± 0·16|
|Leptin, pg/ml||10 503·86 ± 11894·16|
|Soluble interleukin-6 receptor, pg/ml|| 5 542·58 ± 1 819·79|
|Soluble vascular adhesion molecule-1, ng/ml|| 84·18 ± 13·99|
|High-sensitive-C-reactive protein, mg/ml|| 2·40 ± 0·32|
Comparisons of tissue concentrations of parameters between patients with and without diabetes mellitus are given in Table 2. The mean tissue levels of leptin, hs-CRP and sIL-6R were significantly higher in patients with diabetes mellitus than without diabetes mellitus. These differences in the variables between the presence and absence of diabetes mellitus remained significant after adjusting for age, sex, BMI and statin use. However, the mean tissue levels of sVCAM-1 were not found to be statistically significant between the groups according to diabetic state (data not shown). We did not observe any significant differences in any of the inflammatory parameters in relation to the type of medication, hypercholesterolaemia, hypertension, family history of CHD and prior MI (data not shown).
Table 2. Comparisons of tissue concentrations of parameters between patients with (n = 14) and without (n = 21) diabetes mellitus (age, sex, body mass index and statin use adjusted).
|Parameters||Diabetes mellitus (–)||Diabetes mellitus (+) ||t||P|
|mean ± s.e.||95% CI||mean ± s.e.||95% CI|
|Leptin (pg/ml)*||3·43 ± 0·07||3·28–3·57||4·19 ± 0·09||4·02–4·38||28·019||< 0·001|
|sIL-6R (pg/ml)*||3·65 ± 0·02||3·61–3·70||3·83 ± 0·03||3·77–3·89||21·298||< 0·001|
|hs-CRP (mg/ml)||2·25 ± 0·06||2·13–2·37||2·64 ± 0·07||2·49–2·79||17·610||< 0·001|
When patients were classified according to smoking status, the mean tissue levels of leptin, hs-CRP and sIL-6R were significantly higher in current smokers than both former smokers and non-smokers (Table 3). In addition, the mean tissue levels of leptin and sIL-6R were significantly higher in former smokers than non-smokers. These differences remained significant after adjusting for age, sex, BMI and statin use. However, the mean tissue levels of sVCAM-1 were not found to be statistically significant between the groups according to smoking status (data not shown).
Table 3. Comparisons of tissue concentrations of parameters according to cigarette smoking status* (age, sex, body mass index and statin use adjusted).
|Cigarette smoking status (n = 35)||Parameters|
|Leptin** (pg/ml)||sIL-6R** (pg/ml)||hs-CRP (mg/ml)|
|Current smoker||4·37 ± 0·17a||3·88 ± 0·08d||2·98 ± 0·86g|
|Former smoker||3·72 ± 0·30b||3·73 ± 0·04e||2·33 ± 0·04h|
|Non-smoker||3·24 ± 0·11c||3·60 ± 0·08f||2·22 ± 0·03i|
Regression analysis results for leptin, hs-CRP and sIL-6R are given in Table 4. There was a positive association between leptin and hs-CRP, sIL-6R and plasma glucose in all patients. Plasma HDL levels were associated negatively with atherosclerotic tissue levels of leptin. Tissue levels of sIL-6R were associated significantly positively with leptin, hs-CRP and plasma glucose, while tissue levels of hs-CRP were associated with both leptin and sIL-6R.
Table 4. Regression analysis results.
|Parameters||Leptin||sIL-6R||hs-CRP||Plasma glucose||Plasma HDL||F||P||Adj. R2|
|Leptin|| || || ||0·616||5·115||< 0·001||0·478||3·798||0·001||0·088||2·366||0·024||− 0·183||3·079||0·004||1741·224||< 0·001||0·995|
|IL-6R||0·334|| 2·144|| 0·04|| || || ||0·474||3·755||0·001||0·108||2·747||0·01|| || || ||1577·336||< 0·001||0·994|
|hs-CRP||0·782||15·773||< 0·001||0·222||4·472||< 0·001|| || || || || || || || || ||2613·411||< 0·001||0·993|
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- Materials and methods
In the present study, tissue levels of leptin were associated positively with hs-CRP and sIL-6R in atherosclerotic plaques. Our results are in line with previous reports showing that leptin may regulate immune responses, and stimulation of cultured human endothelial cells with leptin may lead to enhanced proinflammatory activity [22–24]. Leptin, either directly or indirectly through the immune system, may alter CRP levels. First, similar to previous reports [23,25], there was a positive association between leptin and both hs-CRP and sIL-6R. Secondly, the long form of the leptin receptor (Ob-R) resembles the gp120 family of cytokine receptors, which includes the IL-6 receptor . Moreover, Ob-R has been shown to have the signalling capabilities of IL-6-type cytokine receptors . Ultimately, the relationship between leptin, IL-6 and CRP in the plaques appears to be more complex. Leptin can stimulate a proinflammatory response, but increased proinflammatory cytokines can also stimulate leptin production. Higher levels of leptin may affect the function and trafficking of inflammatory cells in the atherosclerotic plaques by modulating the production of these cytokines.
Leptin is associated negatively with HDL cholesterol in atherosclerotic plaques. Similar to our results, an inverse correlation between plasma leptin and HDL cholesterol was also observed in some human studies [28,29].
In the current study, the finding of a strong positive association between tissue levels of sIL-6R and hs-CRP was not surprising. Classically, synthesis of CRP in the liver is mainly under the control of IL-6 . Previous studies have demonstrated that CRP is a physiological regulator of sIL-6R shedding in human neutrophils and increases markedly the formation of the sIL-6R/IL-6 complex . Data from a previous experimental study suggest that CRP may also function to increase IL-6 secretion from endothelial cells . Haddy et al. showed that there was a strong correlation between IL-6 and CRP . Therefore, it may be speculated that IL-6 is not only a potent hepatic stimulus for CRP but also associated with increased tissue levels in atherosclerotic plaque.
Cigarette smoking is a well-established cardiovascular risk factor that is also associated with systemic inflammation . Our data suggest that progressively elevated atherosclerotic tissue levels of leptin, hs-CRP and sIL-6R, but not sVCAM-1, were related significantly to smoking status. Furthermore, these findings support previous observations that significantly higher levels of hs-CRP and IL-6 were reported in current smokers than in non-smokers  and smoking influences circulating concentrations of both leptin and CRP [36,37].
In our study, atherosclerotic tissue levels of leptin, hs-CRP and sIL-6R were significantly higher in patients with diabetes mellitus than without diabetes mellitus. In addition, tissue levels of leptin are associated positively with plasma glucose. Consistent with present observations, elevated leptin levels, along with other proinflammatory cytokines, have been associated with an increased risk for type II diabetes [38,39]. Recent evidence suggests that CRP, like leptin, correlates with insulin resistance independently of BMI . It has also been demonstrated that plasma leptin levels are associated with coronary atherosclerosis in type II diabetes . Kado et al. reported that serum levels of IL-6 and the IL-6/IL-6R complex are elevated in diabetic patients . Consequently, our results confirm that leptin, along with other inflammatory markers such as hs-CRP and sIL-6R, may contribute to the progression of atherosclerosis in diabetes mellitus.
Several limitations of the present study deserve mention. The sample size was too small compared to other studies, and the design was too simple. The detection of atherosclerotic tissue levels of other inflammatory markers, which are relevant to the atherosclerotic process, such as tumour necrosis factor-alpha, IL-1beta or chemokines, would have been more convincing. In addition, measurement of tissue IL-6 would have strengthened the study.
In conclusion, it is attractive to speculate that hs-CRP, sIL-6R and leptin could act synergistically in the course of local inflammatory activity and those molecules may not be just markers of inflammation and cardiovascular risk but are also likely to play a pathogenic role in atheromatous plaque. In addition, atherosclerotic tissue levels of CRP, sIL-6R and leptin were significantly higher in current smokers and patients with diabetes. In the current study there was no association between tissue levels of variables and blood lipids. However, based on these observations it is not possible to claim that circulating lipids, which are well known to influence both the initiation and progression of CHD, do not play a role in the pathogenesis of atherosclerosis.