*2.4. LOE Reduces Atherosclerotic Plaque Burden in apoE*−*/*<sup>−</sup> *Mice in WD-Fed apoE*−*/*<sup>−</sup> *Mice*

Hematoxylin and eosin (H&E) staining showed atherosclerotic plaques predominantly in the aortic sinus. The plaque area in the aortas of WD-fed apoE−/<sup>−</sup> mice was increased compared with that of aortas derived from C57BL/6 mice (Figure 5). While losartan failed to reduce the atherosclerotic lesion area in WD-fed apoE−/<sup>−</sup> mice (0.47 <sup>±</sup> 0.07 vs. 0.55 <sup>±</sup> 0.03 mm<sup>2</sup> ), LOE treatment significantly reduced the aortic plaque area (0.33 <sup>±</sup> 0.03 vs. 0.55 <sup>±</sup> 0.03 mm<sup>2</sup> ) (Figure 5).

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**Figure 4.** Effect of LOE and losartan treatments on plaque inflammation in the aortas of apoE<sup>−</sup>/<sup>−</sup> mice. LOE treatment significantly reduced the degree of plaque inflammation in western diet-fed apoE<sup>−</sup>/<sup>−</sup> mice, whereas losartan treatment did not. In vivo imaging of atherosclerotic plaque inflammation was assessed by fluorescence reflectance imaging (FRI) using AP-HGC-Cy5.5 nanoparticles. The extent of plaque inflammation was measured with an IVIS-200 FRI system. (**a**) Middle: FRI; bottom: merged images. (**b**) Corresponding cumulative data. The results are expressed as mean ± SEM (n = 4–6). \* *p* < 0.05 versus the C57BL/6 group and # *p* < 0.05 versus the apoE<sup>−</sup>/<sup>−</sup> vehicle group. **Figure 4.** Effect of LOE and losartan treatments on plaque inflammation in the aortas of apoE−/<sup>−</sup> mice. LOE treatment significantly reduced the degree of plaque inflammation in western diet-fed apoE−/<sup>−</sup> mice, whereas losartan treatment did not. In vivo imaging of atherosclerotic plaque inflammation was assessed by fluorescence reflectance imaging (FRI) using AP-HGC-Cy5.5 nanoparticles. The extent of plaque inflammation was measured with an IVIS-200 FRI system. (**a**) Middle: FRI; bottom: merged images. (**b**) Corresponding cumulative data. The results are expressed as mean ± SEM (*n* = 4–6). \* *p* < 0.05 versus the C57BL/6 group and # *p* < 0.05 versus the apoE−/<sup>−</sup> vehicle group. predominantly in the aortic sinus. The plaque area in the aortas of WD-fed apoE−/− mice was increased compared with that of aortas derived from C57BL/6 mice (Figure 5). While losartan failed to reduce the atherosclerotic lesion area in WD-fed apoE−/− mice (0.47 ± 0.07 vs. 0.55 ± 0.03 mm2), LOE treatment significantly reduced the aortic plaque area (0.33 ± 0.03 vs. 0.55 ± 0.03 mm2) (Figure 5).

**Figure 5.** Effect of LOE and losartan treatments on the atherosclerotic plaque areas of aortic sections obtained from apoE−/<sup>−</sup> mice. LOE treatment significantly reduced the extent of aortic plaque, whereas losartan failed to reduce the area of atherosclerotic lesion in western diet-fed apoE−/<sup>−</sup> mice. The atherosclerotic plaque area (mm<sup>2</sup> ) was measured as the area between the internal elastic lamina and the lumen and quantified by ImageJ after H&E staining. (**a**) H&E staining. Asterisk (\*) indicates atherosclerotic plaque. (**b**) Corresponding cumulative data. The scale bar represents 200 µm. The results are expressed as mean <sup>±</sup> SEM (*<sup>n</sup>* = 4–6). \* *<sup>p</sup>* < 0.05 versus the C57BL/6 group, # *<sup>p</sup>* < 0.05 versus the apoE−/<sup>−</sup> vehicle group and †*p* < 0.05 versus the apoE−/<sup>−</sup> LOE group.

(**a**) (**b**)

### **3. Discussion**

The remnants of apolipoprotein B-containing lipoprotein within the arterial walls [25] and subsequent oxidative modification trigger an inflammatory response and endothelial dysfunction [26,27], leading to the formation of atherosclerotic plaques. Current therapeutics against atherosclerotic cardiovascular diseases cannot completely prevent vascular ROS-induced atherogenesis. The major findings of the present study indicate that LOE decreases vascular oxidative stress by suppressing the expression of NADPH oxidase, resulting in improved endothelial dysfunction and in the prevention of atherosclerotic inflammation and lesion progression in a mouse model of atherosclerosis.

A damaged endothelium disturbs the balance between vasoconstrictors and vasodilators and leads to multiple events that promote or exacerbate atherosclerosis [28]. Several studies have suggested that a number of polyphenol-rich natural products are capable of improving endothelium-dependent relaxation by enhancing the endothelial production of vasoprotective factors including NO and EDH and ultimately prevent endothelial dysfunction in cardiovascular diseases including hypertension [29–31]. Our previous studies revealed that LOE was a potential vasorelaxant acting via NO, and that LOEinduced relaxation was attenuated by PI3-kinase/Akt pathway inhibitors in isolated aortic rings. LOE induced a time-dependent phosphorylation of Akt at Ser473 and eNOS at Ser1177 in endothelial cells [22]. The findings suggest that LOE was an activator of the PI3-kinase/Akt-dependent eNOS phosphorylation at Ser 1177. In addition, LOE treatment attenuated endothelial dysfunction and hypertension induced by angiotensin II in rats [22]. Long-term administration of LOE to diabetic mice restored the abolished endothelium-dependent relaxation to Ach in aortic rings and ameliorated hyperglycemia partially [23]. The current findings based on aortic rings isolated from apoE−/<sup>−</sup> mice, an experimental model of atherosclerosis, clearly indicate that chronic LOE treatment improved endothelium-dependent vascular relaxation to Ach in WD-fed apoE−/<sup>−</sup> mice to a degree comparable to losartan. Losartan is a well-known angiotensin II receptor antagonist with anti-hypertensive activity and is used to treat hypertension and heart failure. Previous studies have shown that losartan exhibits anti-atherosclerotic effects in apoE−/<sup>−</sup> mice by reducing lipid accumulation and macrophage infiltration as well as by inhibiting LDL lipid peroxidation [32,33].

Endothelial dysfunction is triggered by high levels of NADPH oxidase-derived superoxide anions, which react with NO, thereby decreasing its bioavailability in the arterial wall. NADPH oxidase, which was detected in neutrophils, is found in vascular endothelial cells and smooth muscle cells, and essential source of superoxide formation in several animal models of vascular disease [34]. Furthermore, p22phox, one of the NADPH oxidase components, has been identified in atherosclerotic coronary arteries of humans [35]. In this study, we demonstrated that LOE inhibited the increased levels of vascular oxidative stress in apoE−/<sup>−</sup> mice as indicated by the pronounced DHE staining in all of the aortic plaques and the arterial wall as well as the excessive vascular expression of NADPH oxidase subunits, including p22phox and p47phox. The findings indicate that the potential effect of LOE intake on endothelium-dependent vascular relaxation is most likely associated with its ability to reduce vascular oxidative stress, partially via decreasing the NADPH oxidase expression in apoE−/<sup>−</sup> mice. Indeed, previous studies suggested that tea and grape-derived polyphenols downregulate the expression of NADPH oxidase subunits such as p22phox and nox1, and tea polyphenols induce upregulation of catalase expression in vascular cells [29,36]. Since endothelial dysfunction is identified ahead of structural alterations in the arterial wall, it most likely acts as an early signaling event in the pathological initiation and progression of atherosclerosis [28].

Inflammation, which is mediated by various factors such as cytokines, adhesion molecules, and NO, is considered a vital component of atherogenesis [1,3]. Several studies have shown that dietary polyphenols, especially theaflavin and quercetin, ameliorate atherosclerosis by improving inflammation and bioavailability of NO in apoE−/<sup>−</sup> mice [37,38]. We employed molecular FRI as a sensing platform to measure plaque inflammation after injecting AP-HGC-Cy5.5 nanoparticles as molecular imaging agents. These nanoparticles selectively distinguish atherosclerotic plaques by binding to the IL-4 receptor on macrophages, endothelial cells, and smooth muscle cells. Therefore, this molecular imaging tool facilitates the visualization of early atherosclerotic plaque lesions [39]. We detected that increased plaque inflammation in WD-fed apoE−/<sup>−</sup> mice and LOE treatment for 20 weeks resulted in marked regression of plaque inflammation, even more than losartan treatment. A reduction in plaque inflammation in LOE-treated apoE−/<sup>−</sup> mice decreased the aortic plaque area, suggesting anti-atherogenic and anti-inflammatory effects of LOE in apoE−/<sup>−</sup> mice.

### **4. Materials and Methods**
