# Phage Display Preparation of Specific Polypeptides in Atherosclerotic Foam Cells
## 4 Discussion  
Atherosclerosis is a chronic inflammatory disease within vascular walls; the formation of foam cells in a vascular wall is a characteristic feature of atherosclerotic plaques [15]. Monocytes migrate from the circulation into the intima of the arterial wall, where they differentiate into macrophages, which then uptake modified lipoproteins via a relatively large family of scavenger receptors (such as SR-A and CD36), transforming into foam cells [16]. ROS production, such as superoxide, hydrogen peroxide, and peroxynitrite, together with inflammatory factors such as cytokines, chemokines, and adhesion molecules have been shown to be increased in atherosclerotic lesion formation in atherosclerosis [17; 18; 19]. Thus, ROS is undoubtedly a representative indicator of foam cell and lesion formation. In this study, we selected a ROS-specific cell-permeable fluorescent probe 2\({}^{\prime}\),7\({}^{\prime}\)-dichlorofluorescein diacetate (DCFH-DA) to detect the ROS in foam cells. DCFH-DA is a nonpolar dye which is converted into the polar derivative 2\({}^{\prime}\),7\({}^{\prime}\)-dichlorofluorescein (DCFH) by cellular esterase. DCFH is nonfluorescent, very sensitive to several ROS, and can be oxidized to a highly fluorescent 2\({}^{\prime}\),7\({}^{\prime}\)-dichlorofluorescein (DCF) when oxidized by intracellular ROS or other peroxides, having an excitation wavelength of 485 nm and an emission band between 500 and 600 nm [20]. The fluorescence signal was collected and analyzed by spectrophotometer and flow cytometry in our work (Figure 3). The THP-1 and derived foam cells were both treated with 5 \(\upmu\)M and 10 \(\upmu\)M DCFH-DA, respectively. The fluorescence intensity from foam cells is stronger than THP-1 (Figure 3a). In addition, the flow cytometry showed the same results (Figure 3b), i.e., the foam cells' fluorescence intensity (blue curve) is stronger than THP-1 (green curve) on the horizon axis, and 100 times stronger than that of control groups. The ROS detection results show that foam cells have been successfully induced from THP-1. Oil Red O is a fat-soluble diazo dye with maximum absorption at 518 nm, which stains neutral lipids and cholesteryl esters but not biological membranes. The hydrophobic Oil Red O will move from the solvent to associate with the lipid droplets within foam cells [21]. DL receptor (LDLR) is an essential factor for maintaining cholesterol homeostasis. LDLR is a cell-surface receptor that removes the cholesterol-rich LDL from plasma and maintains the cholesterol level in the blood circulation [22]. CD36 functions as a high-affinity receptor for ox-LDL, and binding of ox-LDL to CD36 leads to endocytosis through a lipid raft pathway. Therefore, the pathogenic role of ox-LDL in atherosclerosis largely depends on CD36 [6]. ABCA1 (adenosine triphosphate-binding cassette transporter A1) is a member of the large superfamily of ABC transporters and plays a critical role in the prevention of macrophage foam cell formation and atherosclerosis by mediating the active transport of intracellular cholesterol and phospholipids to apoA-I [23]. Typically, the most widely characterized mechanism by which HDL-C protects against atherosclerosis is reverse cholesterol transport. In this pathway, pivotal steps comprise the ABCA1-mediated cholesterol efflux followed by cholesterol esterification via lecithin-cholesterol acyltransferase [24; 25]. Thus, ABCA1 expression could be regarded as a macrophage response to cholesterol stress, and overexpression could be correlated with foam cell formation. Similarly, ABCG1 (adenosine triphosphate-binding cassette transporter G1) mediates cholesterol removal from macrophages to HDL particles but not lipid-free apoA-I. Thus, the formation of foam cells is mainly due to uncontrolled uptake of LDL or impaired cholesterol efflux in macrophages. In this process, CD36 is responsible for the internalization of modified LDL, promoting cellular accumulation of cholesterol.