Custerin/Apolipoprotein J is a secreted protein biosensor of oxidative stress, which is upregulated in a wide variety of pathological processes including aging, neurodegeneration, diabetes and atherosclerosis [8, 9]. Although the precise function of the molecule is still under investigation, it has been accepted that CLU exerts cytoprotective and anti-inflammatory actions . Additionally to its roles as secreted protein, it has been reported that might act intracellularly regulating homeostasis in human cells .
High serum concentration of CLU has been coupled to vascular damage and generalized stress conditions such as type II diabetes and coronary artery disease  and it has been associated with significant coronary stenosis .
In aortic tissue, CLU expression increases with atherosclerosis progression from fatty streaks to advanced lesions [7, 10] while its presence in normal aortas is unobtrusive . The molecule localization on vascular tissue could be attributed to HDL particles’ penetration from plasma, production through vascular smooth muscle cells (VSMC), release from activated platelets and retention in vascular wall through glycoproteins of extracellular matrix . Recently, it has been shown that CLU expression in VSMC is induced by cellular RNA released from necrotic cells in atherosclerotic lesions through toll-like receptor 3  while it seems that inhibits the apoptosis of VSMC in vitro by binding to modified-LDL particles . The enhanced expression of CLU on injured tissue may serve the clearance of necrotic cell debris contributing to its protective role.
To our knowledge, a few studies enter into CLU and carotid atherosclerosis [12, 13] while apolipoprotein’s expression on human carotid tissue and its association with parameters related to the disease development has not been examined. This was the purpose of the present work where the degree of CLU immunolocalization on carotid artery was tested for its relationship with demographic characteristics, blood parameters and pharmacological treatment of patients underwent internal carotid artery endarterectomy.
A number of parameters including classic biochemical and hematological parameters, thrombosis related parameters and inflammatory factors as well as the percentage of carotid stenosis were measured. Additionally, diabetic, hypertensive and patients with coronary artery disease participated in the study and pharmacological treatment was recorded aiming to find possible strong associations between CLU expression on carotid artery and parameters, which are related to atherosclerotic disease.
The results of the study showed that CLU expression density on carotid tissue was significantly higher in smoking subjects than in non-smoking ones. A large body of evidence confirms the relationship between smoke and atherosclerotic process. Framingham Heart Study conducted in 4255 men and women revealed that smoking is strongly associated with the risk of cerebral infarction . Interestingly, the risk becomes significantly lower two years after smoking cessation while it returns to the risk for non-smokers five years after cessation. The prevalence of clinically significant internal carotid stenosis (≥50%) increases from the percentage of 4.4% in non-smokers to 7.3% in ex-smokers climbing to 9.5% in current smokers . Smoking is thought to cause oxidative damage and is considered as a source of cellular stress. Since bibliographical data supports a role of CLU as a sensitive biomarker of oxidative stress  it is concluded that the higher expression of the molecule on the carotid tissue of smoking subjects reflects oxidative damage and cellular stress.
In the opposite site, molecule expression was significantly lower in patients on clopidogrel treatment and a trend for lower levels in patients taking ASA was observed. Both clopidogrel and ASA are anti-platelet agents that are indicated for the prevention of vascular ischemic events in patients, which are prone to atherosclerotic disease. The CAPRIE study, the bigger study until today where clopidogrel was used as antiplatelet agent, revealed that it is superb (75 mg/day) than ASA (325 mg/day) in secondary stroke prevention .
Since clopidogrel and ASA inhibit platelet aggregation in the endothelium impede atherosclerotic process exerting protective properties in vascular wall and this may explain the lower expression of CLU in carotid tissue. Another parameter, which might implicate in the lower presence of the molecule, is that CLU has been identified as one of the most abundant transcripts of human platelets and is highly expressed on extracellular membrane . Under this point of view anti-platelet agents could inhibit a possible way of CLU localization on vascular tissue.
Another finding of the current study is that serum TNF-a concentration was significantly associated with CLU distribution on carotid artery of the patients and comprises an independent predictor of higher ratio CLU per area. Recent studies demonstrate the role of CLU on repressing NF-κB signaling [22, 23] and it has been supported that interferes with TNF-a causing a negative regulation of inflammatory processes in the vessel wall . The positive association between CLU distribution on carotid artery and serum TNF-a, which was observed in the present study reflects inflammation processes which are taking place during the development of atherosclerosis and possibly indicate the protective role of CLU in the vessel wall since it is known for its anti-proliferative, anti-apoptotic and anti-inflammatory properties . CLU immunolocalization was not associated with the other inflammatory factors, which were measured. This result could be attributed to the small number of participants and the subsequent grate standard deviation, which was observed in these measurements. Additionally, pharmacological treatment influences inflammatory factors’ concentrations, i.e. statins therapy lowers CRP levels, as this was made clear in the JUPITER study .
CLU expression was not higher in diabetic patients or in patients suffering from coronary artery disease. Studies have demonstrated that serum CLU concentration is elevated in these patients’ categories [8, 15] reflecting a generalized stress induction mechanism, which is related to these diseases. Certainly, it was supported that the elevated serum levels indicate vascular damage. A limitation of our study was that serum CLU concentration was not determined in order to reveal its possible association with the molecule immunolocalization on carotid tissue. However, carotid stenosis was not related with CLU tissue expression in our study.
CLU immunolocalization on carotid tissue was expressed as density and as ratio per area. Density refers to the qualitative evaluation of the stain (meaning how “strong” or how “weak” it is). Percentage refers to the positively stained area compared to the whole slide (expressed as % per area). However, further studies using reliable techniques could give explanations related to the different results concerning the association of density and ratio per area with each measured parameter.
Classical biochemical parameters and thrombosis related factors did not show any association with CLU immunolocalization on carotid artery.