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99:0F:35R�>g.�>�5�5�>�5����0��X���s9s9y/���F:++++���������������������������������������������������������������������>���������� �:INCREASED CORONARY ATHEROSCLEROTIC PLAQUE LEVELS OF NEOPTERIN AND C-REACT0VE PROTEIN IN BOTH PATIENTS WITH DIABETES MELLITUS AND SMOKER

Subheading: Increased plaque neopterin and CRP levels
Rahsan I. Sagkan1, Mehmet Karaduman2, Fatih Tangi3, Ugur Musabak1, Celalettin Gunay4, Oben Baysan5, Cagatay Oktenli6*

Department of Immunology and Allergic Diseases1, Intenal Medicine2, Cardiovascular Surgery4, and Cardiology5, G�lhane Military Medical Academy, Ankara, Turkey. Division of Internal Medicine3, G�lhane Military Medical Academy, Haydarpasa Training Hospital, Istanbul, Turkey. Department of Internal Medicine6, Anadolu Medical Center, TR-41400 Gebze/Kocaeli, Turkey.

*Corresponding author
Prof. Cagatay Oktenli, MD.
Department of Internal Medicine, Anadolu Medical Center, TR-41400 Gebze Kocaeli/TURKEY 
E-mail: cagatay.oktenli@anadolusaglik.org or coktenli@yahoo.com
Tel: +90 216 5471122; Fax: +90 216 3179503
Abstract
Backround: Although the inflammatory and immune activation has been assessed extensively by measuring levels in the peripheral blood, limited data is available for the local status of neopterin at the site of the atherosclerotic plaques. Therefore, we aimed to measure the levels of neopterin and high sensitivity-C-reactive protein in the plaques obtained from coronary artery bypass grafting surgery and to evaluate whether there is any relationship between these variables and both clinical and biochemical characteristics. 
Methods: Coronary artery specimens were obtained from 30 consecutive patients (22 men and 8 women) who underwent coronary artery bypass grafting procedure. Sample concentrations of neopterin and high sensitivity-C-reactive protein was measured. 
Results: Coronary atherosclerotic plaque neopterin levels correlated significantly in a positive manner with tissue high sensitivity-C-reactive protein. The mean levels of atherosclerotic plaque neopterin and high sensitivity-C-reactive protein were significantly higher in patients with both smoked in the past and currently smoker patients than patients who never smoked. The mean levels of tissue neopterin and high sensitivity-C-reactive protein were significantly higher in patients with diabetes mellitus than patients without diabetes mellitus. 
Conclusions: Our results provides confirmatory data to prior studies dealing with both neopterin and high sensitivity-C-reactive protein play an important role in pathogenesis of coronary atherosclerotic plaque in both status of diabetes and smoking. However, as either neopterin or high sensitivity-C-reactive protein is only one of the intrinsic factors in the inflammatory process of coronary atherosclerotic plaque, other inflammatory markers in the process needs to be investigated further.
Key Words: C-reactive protein, coronary atherosclerotic plaque, diabetes, neopterin, smoking
Introduction

Recent studies in patients with atherosclerotic heart disease (AHD) show inflammation plays a crucial role in both pathogenesis and progression of the disease and its presentation as acute coronary syndromes [1-3]. In particular, it is well known that the monocyte-macrophage system, T lymphocytes and proinflammatory cytokines are involved in the pathogenesis of atherosclerotic plaque and its thrombotic complications [4,5]. In this way, coronary atherosclerotic plaque may be considered as a inflammatory nidus that is filled with the immune cells that can orchestrate and effect many inflammatory processes. Neopterin, a pteridine derivative and a byproduct of the guanosine triphosphate-biopterin pathway, is produced by interferon-�-stimulated macrophages. In practice, neopterin is used as a soluble marker for cellular immune response and macrophage activity [6]. Moreover, it has been suggested that neopterin play a pivotal role in pathogenesis of atheromatous plaque disruption via activation of both constitutive and inductible nitric oxide synthase, and promoting the expression of proinflammatory genes [7,8]. Accumulating evidence suggested neopterin levels were high in patients with AHD and associated with its severity, the complexity of atherosclerotic lesions and an increase in cardiovascular risk [9-14]. On the other hand, C-reactive protein (CRP) also plays an intrinsic role in pathogenesis of coronary events due to atherosclerosis [1,15,16]. Mazer et al. suggested that CRP produced locally in atherosclerotic plaques by resident macrophages and vascular smooth muscle cells and it may be involved in several steps in plaques genesis and progression [17]. In atherosclerotic plaque obtained from autopsy samples, it has been demonstrated that CRP mRNA levels are increased [18]. Previously, the gene for CRP was found to be expressed in coronary plaque tissue obtained during atherectomy procedure [19].
Although the inflammatory and immune activation has been assessed extensively by measuring levels in the peripheral blood, limited data is available for the local status of neopterin at the site of the atherosclerotic plaques. Atherectomy specimens obtained from coronary artery bypass grafting (CABG) not only provide a unique source of plaque tissues but also they make it possible to correlate features of plaque biology with the clinical and the biochemical outcomes of the patients [1,2]. Therefore, we aimed to measure the levels of neopterin and high sensitivity-CRP (hs-CRP) in the plaques obtained from CABG surgery and to evaluate whether there is any relationship between these variables and both clinical and biochemical characteristics.











Methods

Thirty consecutive patients (22 men and 8 women) who underwent CABG procedure in Cardiovascular Surgery Department of G�lhane Military Medical Academy Hospital were included to the study. Coronary artery specimens were obtained from the patients during the procedure. Each subjects gave his informed consent to the study, which was previously approved by our local ethics committee and institutional review board. Patients who have prior myocardial infarction within 30 days before surgery, any systemic disease besides atherosclerosis, coexisting neoplasia, immunologic disease, acute or chronic infectious diseases, secondary or familial dyslipidemia, dysfunction of the thyroid gland, impaired liver or kidney function, excessive alcohol consumption, any medication other than acetylsalicylic acid (d" 250 mg/day), nitrates, beta-blocker, calcium antagonists, angiotensin-converting enzyme inhibitors, angiotensin-II receptor blockers, statins or oral antidiabetics were excluded.
	Clinical history was assessed for smoking, hypertension, diabetes mellitus, hyperlipidemia. Smoking was classified as current smoking (smoking more than 5 cigarettes within the past 3 months), smoked in the past (> 3 months and < 40 years), or never smoking. Body mass index was calculated as weight (kilograms) divided by height (meters) squared. Blood pressure was calculated as the average of three measurements taken under standardized conditions in a supine position with a sphygmomanometer.
	Blood samples for laboratory assays were obtained before the procedure following overnight fasting. Serum was separated and frozen at - 80�C until the time of the assay. Fasting blood glucose, triglyceride and high-density lipoprotein levels were measured by enzymatic colorimetric method with Olympus AU 600 autoanalyzer using reagents from Olympus Corp. (Hamburg, Germany). Low-density lipoprotein was calculated by Friedewald�s formula.
	Thirty coronary atherosclerotic plaque specimens were derived from proximal lesions in the left anterior descending coronary artery (n=27), right coronary artery (n=1), left circumflex artery (n=2) during the elective CABG surgery. Immediately after the procedure, all extracted atherosclerotic plaque tissues were frozen and stored at - 80�C until the tissue homogenization.
	The frozen plaques (mean tissue weight, 0.020 g � 0.001) were homogenized in ice cold homogenization buffer (50 mM HEPES, 0.2% Triton X-100, 1 mM EDTA and 0.1 mM PMSF, Ph 7.4) at 13.500 rpm in Ultraturrax T25 (Janke&Kunkel, IKA Labortechnik, Staufen, Germany) using a method that was previously described [20]. The homogenates were centrifuged at 4500 rpm for 15 minutes. Supernatans were coded to ensure anonymity and all analyses were performed in a blinded fashion. Tissue contents of neopterin (IBL International, Hamburg, Germany) were measured by enzyme immunoassay. Absorbance readings were carried out on Alisei Quality System (SEAC Radim Group, Pomezia, Italy). Sample concentrations of neopterin was determined from a curve obtained with the standards. hs-CRP was measured with a human CRP kit from Generic Assays GmbH (Dahlewitz, Germany). The analytical sensitivity of the human CRP kit was determined at 0.2 mg/ml. The intra- and inter-assay CV for hs-CRP were < 5.7% and < 13.6%.



Statistical analysis
	
All statistical analyses were performed by using a package program SPSS 15.0 (SPSSFW, SPSS Inc., Chicago, IL, USA) statistical package. Descriptive statistics were given as arythmetic mean � standard deviation notation. Comparisons between the groups were made by Mann-Whitney U test. Correlations among the parameters were investigated by Spearman rank correlation coefficient (�  Rho-) and Pearson correlation procedure. P values less than or equal to 0.05 were evaluated as statistically significant.












Results
	
Clinical and laboratory characteristics of patients were shown in Table I. Hyperlipidemia were present in 12 (40%) patients, hypertension in 15 (50%), diabetes mellitus in 10 (34%), family history of AHD in 11 (37%), prior MI in 10 (34%). Drug use as follows: aspirin (n=30), beta-blocker (n=17), calcium antagonists (n=14), angiotensin-converting enzyme inhibitors/angiotensin-II receptor blockers (n=28), oral antidiabetics (n=10), nitrates (n=9), and statins (n=24). Smoking status was as follows: current smoking (n=7), smoked in the past (n=12), or never smoking (n=11).
The mean levels of atherosclerotic plaque neopterin and hs-CRP were significantly higher in patients with both former and currently smoker patients than patients who never smoked (Table II). When patients were classified according to the diabetes status, the mean levels of tissue neopterin and hs-CRP were significantly higher in patients with diabetes mellitus than patients without diabetes mellitus (Table II). In addition, no significant differences were found between the variables in terms of hyperlipidemia, hypertension, prior MI, family history of AHD, and medications (data not shown).
Coronary atherosclerotic plaque neopterin levels correlated significantly in a positive manner with tissue hs-CRP (� = 0.946, P < 0.001). There were no association between tissue levels of variables and blood lipids or glucose (data not shown).



Discussion
	
Despite the accumaliting data that neopterin and CRP play an crucial role in pathogenesis of coronary atherosclerosis [1,13-16], little is known about relation between local levels of neopterin and CRP in atherosclerotic plaque. In an immunohistochemical study using coronary atherectomy specimens, it has been firstly demonstrated that a significantly higher prevalance of neopterin-positive macrophages in culprit lesions in patients with unstable angina pectoris than in those with stable angina pectoris [21]. In the current study, we demonstrated that there is a positive correlation between tissue levels of neopterin and hs-CRP in coronary atherosclerotic plaque contents. Our findings support the notion that both neopterin and CRP is not only a marker of AHD activity but they may also play a pathogenic role in physiopathology of the plaques [15,22,23].
	The another important finding of our study is increased tissue levels of neopterin and hs-CRP in coronary atherosclerotic plaques related with both smoking status and diabetic state. It is well known that both status of diabetes and smoking are pro-atherogenic factors. In this way, tissue levels of hs-CRP in coronary atherosclerotic plaques were related with both diabetic state and smoking status in our previous study1. In agreement with these results, both neopterin and CRP were reported to be independent predictors of fatal coronary events in patients with diabetes mellitus [24]. Overall, these data are also in line with the previous observations that diabetes mellitus accelerates the atherosclerotic process by increasing inflammatory processes [25,26]. On the other hand, it is well know that cigarette smoking aggravates the progression of AHD1 and it is also associated with systemic inflammation [27]. In this way, our findings support the previous observations that significantly higher levels of hs-CRP were reported in current smokers when compared the non-smokers [28] and smoking influences circulating concentrations of CRP [29]. Consequently, it seems likely that neopterin and hs-CRP may contribute to the progression of coronary atherosclerosis in both diabetes mellitus and smoking.
	In the current study, there were no association between tissue levels of neopterin and blood lipids. Likewise, in a previous study, lipid lowering therapy with statins did not result in lower levels of neopterin [30]. This leads us to believe that the pathogenic role of neopterin in progression of atherosclerotic plaque are independent of blood lipids.
	Regarding study limitations, we should acknowledge that the localization or origins of neopterin and hs-CRP in atherosclerotic plaque are unclear. Therefore, the localization of these markers immunohistochemically in combination with the staining of cell-specific markers using several specimens would be much more convincing as previously stated [2]. Secondly, our patients were on drugs at least until CABG procedure, so that these medications may create a confounding effects on our results. Another limitation of the current study was the small sample size of females within the study group. However, it is possible that this reflects the relatively smaller representation of females in overall population of patients with AHD. Interestingly, there were no relation between tissue levels of these variables and blood lipids. But, it is impossible to speculate based on our results that circulating lipids, which is well known to influence the progression of coronary artery disease, do not play a role in the pathogenesis of coronary atherosclerosis in both diabetic state and smoking. Finally, because the present study was performed in AHD patients without a reference population, it cannot be readily generalized to non-atherosclerotic individuals.
Our results provides confirmatory data to prior studies dealing with both neopterin and CRP play an important role in pathogenesis of coronary atherosclerotic plaque in both status of diabetes and smoking. However, as either neopterin or CRP is only one of the intrinsic factors in the inflammatory process of coronary atherosclerotic plaque, other inflammatory markers in the process needs to be investigated further.


Disclosure Statement
	Authors of this study have no interest which might be perceived as posing a conflict or bias.


Acknowledgements
This study was supported by grant from the Research Centre of G�lhane Military Medical Academy.







References
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MCJOJQJaJh�+hqU�CJOJQJaJUh�+CJOJQJaJh'�CJOJQJaJh�u}CJOJQJaJhW}�CJOJQJaJh'�hqU�CJOJQJaJh�phqU�CJOJQJaJ"Pradhan AD, Manson JE, Rifai N, Buring JE, Ridker PM. (2001) C-reactive protein, interleukin-6, and risk of developing type 2 diabetes mellitus. J Am Med Assoc 286: 327-334.
Huxley R, Barzi R, Woodward M. (2006) Excess risk of fatal coronary heart disease associated with diabetes in men and women: meta-analysis of 37 prospective cohort studies. Br Med J 332: 73-78.
de Maat MP, Pietersma A, Kofflard M, Sluiter W, Kluft C. (1996) Association of plasma fibrinogen levels with coronary artery disease, smoking and inflammatory markers. Atherosclerosis 121: 185-191.
Bermudez EA, Rifai N, Buring JE, Manson JE, Ridker PM. (2002) Relation between markers of systemic vascular inflammation and smoking in women. Am J Cardiol 89: 1117-1119.
Bazzano LA, He J, Muntner P, Vupputuri S, Whelton PK. (2003) Relationship between cigarette smoking and novel risk factors for cardiovascular disease in the United States. Ann Intern Med 138: 891-897.
Mulder DJ, van Haelst PL, Wobbes MH, Gans RO, Zijlstra F, et al. (2007) The effects of aggressive versus conventional lipid-lowering therap on markers of inflammatory and oxidative stress. Cardiovasc Drugs Ther 21: 91-97.

















Legends of tables

Table 1. Clinical and Laboratory Characteristics of Patients (n = 30)
Table 2. Comparison of coronary atherosclerotic plaque neopterin and hs-CRP levels according to smoking status and diabetes


















 PAGE   \* MERGEFORMAT 11




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