Is plasma-soluble CD36 associated with density of atheromatous plaque and ankle–brachial index in early-onset coronary artery disease patients?

Monika Rać1, Andrzej Krzystolik2, Michał Rać3, Krzysztof Safranow1, Violetta Dziedziejko1, Marta Goschorska1, Wojciech Poncyljusz4, Dariusz Chlubek1

1Department of Biochemistry and Medical Chemistry, Pomeranian Medical University, Szczecin, Poland
2Department of Cardiology, Regional Hospital in Szczecin, Szczecin, Poland
3Department of Diagnostic Imaging and Interventional Radiology, Pomeranian Medical University, Szczecin, Poland
4Department of Interventional Radiology, Pomeranian Medical University, Szczecin, Poland

Address for correspondence:
Monika Rać, PhD, Department of Biochemistry and Medical Chemistry, Pomeranian Medical University, Al. Powstańców Wielkopolskich 72, 70–111 Szczecin, Poland, tel: +48 91 466 15 30, fax: +48 91 466 15 16, e-mail: carmon12@gmail.com
Received: 08.04.2015 Accepted: 22.10.2015 Available as AoP: 27.11.2015

INTRODUCTION

CD36 is a major macrophage scavenger receptor for oxidised low-density lipoprotein (LDL) particles [1] and is believed to play a critical role in the initiation and progression of atherosclerosis through its ability to bind and internalise modified LDL, facilitating formation of lipid-engorged macrophage foam cells [2–4]. In addition, the 7q chromosome region, containing the CD36 gene, has been linked to components of metabolic syndrome in several genome-wide association studies [5, 6]. It has been shown [7, 8] that soluble CD36 (sCD36) is circulating as ligand-bound complex and may be present as an unbound protein or a peptide fraction thereof, or may be present in microparticles shed from cells such as platelets, monocytes/macrophages, or adipocytes after being triggered by various stimuli. Tontonoz et al. [9] hypothesised that decreased sCD36 level is a result of lower activity of endogenous peroxisome proliferator-activated receptor gamma (PPARγ). Plasma CD36 is released into circulation during cell apoptosis, such as that taking place after cholesterol accumulation in foam cells [10, 11]. Positive association of plasma sCD36 concentration with insulin resistance has been reported, and it has been proposed that sCD36 might represent a marker of macrophage activation and inflammation leading to atherosclerosis [12, 13].

We have recently identified an association between CD36 polymorphism and low thickness of atheromatous plaque, suggesting its protective effect against atherosclerosis development [14]. The aim of this study was to obtain insight into the relationship between plasma concentration of sCD36 and radiological parameters of atherosclerosis in patients with early-onset coronary artery disease (CAD).

METHODS

The study group comprised 70 clinically stable patients (18 women and 52 men) with early-onset CAD (men less than 50 years old and women less than 55 years old; age of all patients 49 ± 6 years). All patients were Polish residents hospitalised in the Department of Cardiology of the County Hospital in Szczecin (north-western Poland) and then referred to the outpatient clinic in the years 2008–2011. The study complies with the principles outlined in the Declaration of Helsinki and was approved by our institutional Ethics Committee. Informed consent was obtained from each patient. The criteria for early-onset CAD diagnosis and characteristics of the study group were presented in our previous study [14]. Fasting blood sample was taken for serum glucose, lipid profile (total, high-density lipoprotein [HDL] and LDL cholesterol, and triglycerides), ApoA1, ApoB, Lp(a), and plasma sCD36 protein measurements. In addition, the weight, height, waist and hip circumference, and systolic and diastolic blood pressure of each patient were measured, and the body mass index, waist-to-hip ratio, and mean arterial pressure calculated. Doppler ultrasound examinations of carotid and peripheral arteries were performed in all patients. Thickness of the intima–media complex (IMC) of the common carotid artery (CCA) and brachial artery, and the density and thickness of atheromatous plaque at CCA bifurcation were measured with M’Ath programme [15]. Density measurement was based on the intensity of reflection from the plaque dependent on calcium deposits. The ankle–brachial index (ABI) was calculated by dividing the systolic blood pressure (SBP) in the arteries at the left and right ankle (posterior tibial artery) or foot (anterior tibial artery) by the higher of the two SBPs in the arms.

Plasma concentrations of human CD36 antigen (Platelet Membrane Glycoprotein IV) were measured using commercially available enzyme-linked immunosorbent assay (ELISA) kits (EIAab, Wuhan EIAab Science Co., Ltd., China) according to the manufacturer’s protocol, as previously published by Krzystolik et al. [16].

Correlations between quantitative variables and sCD36 plasma concentration were assessed with the Spearman rank correlation coefficient (Rs). Associations between qualitative variables and sCD36 plasma concentration were tested with the Mann-Whitney U test. P-values < 0.05 were considered statistically significant.

RESULTS

The clinical data of the study group are presented in Table 1. Significantly higher soluble CD36 plasma concentrations were found in females. Therefore, all CAD patients were divided into subgroups of men and women for further analysis. Correlations between quantitative variables and CD36 plasma concentration assessed with the Spearman rank correlation coefficient are presented in Table 2. Associations between qualitative variables and CD36 plasma concentration tested with the Mann-Whitney U test are presented in Table 3. There was no association between concentrations of sCD36 and medication or revascularisation treatment, nor with the presence of past myocardial infarction (MI), hypertension, or type 2 diabetes (data not shown).

Table 1. Characteristics of the study group (n = 70)

Table 2. Correlations between circulating soluble CD36 concentration and quantitative parameters of early-onset coronary artery disease (CAD) patients in the whole study group and in the subgroups of CAD males and females

Table 3. Associations between circulating soluble CD36 concentration and qualitative parameters of early-onset coronary artery disease (CAD) patients in the whole study group and in the subgroups of CAD males and females

There were no significant correlations between sCD36 and radiological parameters of atherosclerosis progress in our patients. We observed only weak negative correlation of sCD36 level with IMC of left brachial artery with borderline significance in the whole group. There was also a significant negative correlation with CCA plaque density, but only in the female subgroup and only on the right side. We found borderline higher sCD36 plasma concentrations in patients with lower ABI value (< 0.9).

DISCUSSION

Increased risk of atherosclerosis is associated with lower ABI and higher thickness of IMC [17]. Density of plaque reflects plaque calcification. Lower plaque calcification is associated with plaque instability, but high plaque calcification is associated with predisposition to thrombosis [18, 19]. We studied radiological parameters of atheromatous plaque in an early-onset CAD group. So far, there has been no publication indicating an association between sCD36 and multiple radiological indicators of atherosclerosis in any population. In the current study we found only borderline association of lower ABI value (< 0.9) with higher sCD36 plasma concentrations. We observed significant negative correlation with CCA plaque density, but only in the female subgroup and only on the right side. On the other hand, we observed only weak negative correlation of sCD36 with IMC of the left brachial artery, and it was not significant. The borderline association of higher sCD36 with lower ABI seems to be advantageous, but on the other hand, the association with lower density of plaques may be disadvantageous. Therefore, there is a lack of clear answers on the relationship between plasma concentration of sCD36 and radiological parameters of atherosclerosis in patients with early-onset CAD. Only one previous study demonstrated results similar to ours [20], showing that Caucasian patients with echolucent carotid plaques tended to have higher soluble CD36 levels compared to those with echogenic/heterogenic plaque. However, the association observed in the cited study was not statistically significant (p = 0.087). Moreover, in that publication plasma sCD36 concentration was measured also by enzyme-linked immunoassay, and this evaluation was performed in patients with high-grade internal carotid stenosis (> 70%) treated with carotid angioplasty with stenting.

In our previous study [14] two CD36 gene polymorphisms were associated with low thickness of atheromatous plaque, suggesting its protective effect against development of atherosclerosis. It is known [21] that CD36 can be detected in atherosclerotic plaques in human aorta and coronary arteries. Its level might be regulated differently with the progress of atherosclerosis in a signalling pathway-coupled cellular uptake of oxidised-LDLs with transcriptional activation of CD36 receptor by PPARγ [9]. In our previous study [16] the higher sCD36 plasma concentrations were significantly positively correlated with HDL-cholesterol and ApoA1 concentrations. We have recently identified [22] an association of CD36 gene rs3173798 polymorphism with younger age of MI and presence of type 2 diabetes. In the present study there were no differences in sCD36 plasma concentrations between patients with present and absent MI or type 2 diabetes. Moreover, we have shown in another study [16] that there was no association between CD36 genotypes and sCD36 concentrations.

In fact, our results, which showed no association between concentrations of sCD36 and MI, hypertension, or type 2 diabetes, are not different from the results obtained by other authors. None of the previous studies showed association of sCD36 with diabetes, MI, or hypertension in a direct comparison of groups with and without the disease. Knøsgaard et al. [23] demonstrated a significant association between sCD36 and fasting plasma glucose, but only in a specific population of morbidly obese individuals. Handberg et al. [24] observed positive correlation between sCD36 and fasting plasma glucose only in a subgroup of subjects with high plasma sCD36 concentration, and this correlation lost statistical significance in multivariate analysis. Liani et al. [25] found significantly higher sCD36 levels in patients with type 2 diabetes (aged 64.1 ± 8.7 years) than in healthy subjects (aged 39 ± 12.8 years), but the groups differed in age, while our previous study [16] revealed that older age is a significant independent predictor of higher plasma sCD36.

We also found significantly higher sCD36 plasma concentrations in the female subgroup of CAD patients. Contrary to our results, other authors [24] reported that in clinically healthy Caucasians plasma sCD36 was significantly lower in women than in men, while Chmielewski et al. [26] reported that serum sCD36 concentration is not related to gender in Caucasian chronic kidney disease patients.

It should be noted that the range of plasma sCD36 concentrations measured in our early-onset CAD patients was high. Similarly to our study, high circulating CD36 levels (up to 22.9 µg/mL) measured by ELISA in patients with type 2 diabetes were reported by Alkhatatbeh et al. [27]. Contrary to our results, Lykkeboe et al. [28] reported plasma sCD36 in healthy Caucasian subjects in the range of 0.05–250 ng/mL. In addition, significant differences in plasma sCD36 concentrations measured by two commercial ELISA sCD36 assays were also reported in the same study [28]. In our opinion the analysis of sCD36 is currently hampered by the lack of widely accepted standardised methods. This limitation may constitute a major cause of inconsistent results of different studies concerning sCD36.

CONCLUSIONS

In summary, the present study has some limitations, such as lack of a control group without early-onset CAD and a relatively low number of cases. The lack of strong associations does not resolve doubts about the relationship between sCD36 and radiological parameters of atherosclerosis progress. Further research is necessary to assess the association of high plasma sCD36 concentrations with the risk of plaque instability. These results should be confirmed in a larger group of patients.

Conflict of interest: none declared

References

1. 1. Nakagawa T, Nozaki S, Nishida M et al. Oxidized LDL increases and interferon-gamma decreases expression of CD36 in human monocyte-derived macrophages. Arterioscler Thromb Vasc Biol, 1998; 18: 1350–1357. doi: 10.1161/01.ATV.18.8.1350.
2. 2. Huh HY, Pearce SFA, Yesner LM et al. Regulated expression of CD36 during monocyte to macrophage differentiation: potential role of CD36 in foam cell formation. Blood, 1996; 87: 2020–2028.
3. 3. Collot-Teixeira S, Martin J, McDermott-Roe C et al. CD36 and macrophages in atherosclerosis. Cardiovasc Res, 2007; 75: 468–477. doi: 10.1016/j.cardiores.2007.03.010.
4. 4. Tuomisto TT, Riekkinen MS, Viita H et al. Analysis of gene and protein expression during monocyte–macrophage differentiation and cholesterol loading-cDNA and protein array study. Atherosclerosis, 2005; 180: 283–291. doi: 10.1016/j.atherosclerosis.2004.12.023.
5. 5. An P, Freedman BI, Hanis CL et al. Genome-wide linkage scans for fasting glucose, insulin, and insulin resistance in the National Heart, Lung, and Blood Institute Family Blood Pressure Program: evidence of linkages to chromosome 7q36 and 19q13 from meta-analysis. Diabetes, 2005; 54: 909–914. doi: 10.2337/diabetes.54.3.909.
6. 6. Malhotra A, Elbein SC, Ng MC et al. Meta-analysis of genome-wide linkage studies of quantitative lipid traits in families ascertained for type 2 diabetes. Diabetes, 2007; 56: 890–896. doi: 10.2337/db06-1057.
7. 7. Alkhatatbeh MJ, Mhaidat NM, Enjeti AK et al. The putative diabetic plasma marker, soluble CD36, is non-cleaved, non-soluble and entirely associated with microparticles. J Thromb Haemost, 2011; 9: 844–851. doi: 10.1111/j.1538-7836.2011.04220.x.
8. 8. Koonen DP, Jensen MK, Handberg A. Soluble CD36- a marker of the (pathophysiological) role of CD36 in the metabolic syndrome? Arch Physiol Biochem, 2011; 117: 57–63. doi: 10.3109/13813455.2010.543136.
9. 9. Tontonoz P, Nagy L, Alvarez JG et al. PPARgamma promotes monocyte/macrophage differentiation and uptake of oxidized LDL. Cell, 1998; 93: 241–52. doi:10.1016/S0092-8674(00)81575-5.
10. 10. Glintborg D, Højlund K, Andersen M et al. Soluble CD36 and risk markers of insulin resistance and atherosclerosis are elevated in polycystic ovary syndrome and significantly reduced during pioglitazone treatment. Diabetes Care, 2008; 31: 328–334. doi: 10.2337/dc07-1424.
11. 11. Handberg A, Norberg M, Stenlund H et al. Soluble CD36 (sCD36) clusters with markers of insulin resistance, and high sCD36 is associated with increased type 2 diabetes risk. J Clin Endocrinol Metab, 2010; 95: 1939–1946. doi: 10.1210/jc.2009-2002.
12. 12. Fernández-Real JM, Handberg A, Ortega F et al. Circulating soluble CD36 is a novel marker of liver injury in subjects with altered glucose tolerance. J Nutr Biochem, 2009; 20: 477–484. doi: 10.1016/j.jnutbio.2008.05.009.
13. 13. Handberg A., Levin K, Hojlund K, Beck-Nielsen H. Identification of the oxidized low-density lipoprotein scavenger receptor CD36 in plasma: a novel marker of insulin resistance. Circulation, 2006; 114: 1169–1176. doi: 10.1161/CIRCULATIONAHA.106.626135.
14. 14. Rać ME, Safranow K, Rać M et al. CD36 gene is associated with thickness of atheromatous plaque and ankle-brachial index in patients with early coronary artery disease. Kardiol Pol, 2012; 70: 918–923.
15. 15. Touboul PJ, Hennerici MG, Meairs S et al. Mannheim carotid intima–media thickness consensus (2004–2006). An update on behalf of the Advisory Board of the 3rd and 4th Watching the Risk Symposium, 13th and 15th European Stroke Conferences, Mannheim, Germany, 2004, and Brussels, Belgium, 2006. Cerebrovasc Dis, 2007; 23: 75–80. doi: 10.1159/000097034.
16. 16. Krzystolik A, Dziedziejko V, Safranow K et al. Is plasma soluble CD36 associated with cardiovascular risk factors in early onset coronary artery disease patients? Scand J Clin Lab Invest, 2015; 75: 398–406. doi: 10.3109/00365513.2015.1031693.
17. 17. Weatherley BD, Nelson JJ, Heiss G et al. The association of the ankle-brachial index with incident coronary heart disease: the Atherosclerosis Risk In Communities (ARIC) study, 1987–2001. BMC Cardiovasc Disord, 2007; 7: 3. doi: 10.1186/1471-2261-7-3.
18. 18. Alexopoulos N, Raggi P. Calcification in atherosclerosis. Nat Rev Cardiol, 2009; 6: 681–688. doi: 10.1038/nrcardio.2009.165.
19. 19. Shaalan WE, Cheng H, Gewertz B et al. Degree of carotid plaque calcification in relation to symptomatic outcome and plaque inflammation. J Vasc Surg, 2004; 40: 262–269. doi:10.1016/j.jvs.2004.04.025.
20. 20. Handberg A, Skjelland M, Michelsen AE et al. Soluble CD36 in plasma is increased in patients with symptomatic atherosclerotic carotid plaques and is related to plaque instability. Stroke, 2008; 39: 3092–3095. doi: 10.1161/STROKEAHA.108.517128.
21. 21. Nakagawa-Toyama Y, Yamashita S, Miyagawa J et al. Localization of CD36 and scavenger receptor class A in human coronary arteries: a possible difference in the contribution of both receptors to plaque formation. Atherosclerosis, 2001; 156: 297–305. doi: 10.1016/S0021-9150(00)00662-6.
22. 22. Rać ME, Suchy J, Kurzawski G et al. Polymorphism of the CD36 gene and cardiovascular risk factors in patients with coronary artery disease manifested at a young age. Biochem Genet, 2012; 50: 103–111. doi: 10.1007/s10528-011-9475-z.
23. 23. Knøsgaard L, Thomsen SB, Støckel M et al. Circulating sCD36 is associated with unhealthy fat distribution and elevated circulating triglycerides in morbidly obese individuals. Nutr Diabetes, 2014; 4: e114. doi: 10.1038/nutd.2014.11.
24. 24. Handberg A. Plasma sCD36 is associated with markers of atherosclerosis, insulin resistance and fatty liver in a nondiabetic healthy population. J Intern Med, 2011; 271: 294–304. doi: 10.1111/j.1365-2796.2011.02442.x.
25. 25. Liani R, Halvorsen B, Sestili S et al. Plasma levels of soluble CD36, platelet activation, inflammation, and oxidative stress are increased in type 2 diabetic patients. Free Radic Biol Med, 2012; 52: 1318–1324. doi: 10.1016/j.freeradbiomed.2012.02.012.
26. 26. Chmielewski M, Bragfors-Helin AC, Stenvinkel P et al. Serum soluble CD36, assessed by a novel monoclonal antibody-based sandwich ELISA, predicts cardiovascular mortality in dialysis patients. Clin Chim Acta, 2010; 411: 2079–2082. doi: 10.1016/j.cca.2010.09.009.
27. 27. Alkhatatbeh MJ, Enjeti AK, Acharya S et al. The origin of circulating CD36 in type 2 diabetes. Nutr Diabetes, 2013; 3: e59. doi: 10.1038/nutd.2013.1.
28. 28. Lykkeboe S, Larsen AL, Handberg A. Lack of consistency between two commercial ELISAs and against an in-house ELISA for the detection of CD36 in human plasma. Clin Chem Lab Med, 2012; 50: 1071–1074. doi: 10.1515/cclm-2011-0950.