3.2.2. Inflammation and Atherosclerosis

Atherosclerosis is an inflammatory disease that is driven by endothelial dysfunction. Many patients do not have symptoms for a long time until a plaque ruptures or severe narrowing or total blocking of the blood vessel occurs. Although there are biomarkers to identify atherosclerosis [115,116], tools that can predict the growth dynamics of a plaque or its risk to rupture are hardly available. CEUS with targeted MB could suit this purpose considering its high sensitivity and capability to quantify even low amounts of the inflammation-related markers. Vascular cell adhesion molecule 1 (VCAM-1) is known to be overexpressed and translocated to the luminal surface of endothelial cells at the early stages of atherosclerosis [117,118]. Thus, VCAM-1-targeted MB have been proposed for atherosclerosis imaging [63–71,73,74]. These UCA have successfully identified atherosclerosis and the CEUS signals could be correlated to the progression of the disease [65,67]. Moreover, molecular changes during apocynin [63,64,69] and statin [66] therapy were successfully monitored. Further, VCAM-1, platelet glycoprotein (GP) Ibα [63,64,69], junctional adhesion molecule A (JAM-A) [62], GP VI [94], lectin-type oxidized low-density lipoprotein receptor 1 (LOX-1) [74], and von Willebrand factor (VWF) [69,74] have been used for atherosclerosis imaging. Of these markers, JAM-A expression might be particularly associated with early plaque formation and vulnerability [119]. Indeed, in mice with partial ligation and atherogenic diet molecular US of JAM-A indicated atherosclerosis at a very early stage and was even capable of assessing vascular sites that underwent an immediate change in shear stress [61]. Moreover, another group demonstrated that plaque vulnerability can be assessed in rabbits [62]. Another interesting target is GP VI since it can be used for atherosclerosis therapy. It has been shown that administering soluble anti-GP VI antibody inhibits thrombus formation and progression of atherosclerosis [120–122]. Using high-frequency US, it is possible to disrupt MB and release the ligand coupled to the shell. Thus, GP VI-targeted MB could work as theranostic agents. A study showed that these MB help to diagnose atherosclerosis, and in combination with the high-frequency US work as a therapy [94].

Another pathology that would benefit from a broadly available reliable non-invasive detection technique is acute cardiac ischemia. Electrocardiogram and serological markers often have limited expressiveness or lead to misjudgment [123–126]. The gold standard in the clinics is cardiac magnetic resonance imaging (MRI) though it is time-consuming, requires multiple breath holds, and due to usage of gadolinium cannot be used in patients with renal dysfunction. Other diagnosis methods include scintigraphy and coronary CT. However, both techniques raise safety concerns due to usage of radioisotopes and/or radiation. Myocardial ischemia is associated with endothelial upregulation of adhesion molecules which persist after ischemia has resolved. Thus, it could be possible to identify post-ischemic myocardium using the molecular US. P-selectin seems to be a good target since it is expressed within minutes after ischemia or injury [127]. As expected, P-selectin-targeted MB strongly bound in post-ischemic myocardium in mice [74,83]. Similar results were also seen for E-selectin-targeted UCA in rats (Figure 6) [88]. Moreover, the researchers decided to target both, P- and E-selectins [84,85,128,129]. This proved to be particularly useful since P-selectin was present immediately after ischemia but only E-selectin was still overexpressed after 24 h [84]. Thus, the dual-targeted MB could detect myocardial ischemia for a longer time. Further in vivo experiments were performed in primates demonstrating that molecular US using selectin-targeted MB is a safe and effective method to detect myocardial ischemia [129]. However, despite these promising results, molecular US imaging of the heart is difficult to perform, user-dependent, and requires an experienced physician. Thus, it must be carefully evaluated whether a high diagnostic accuracy and reproducibility can also be achieved outside specialized centers and whether it can compete with the clinically established imaging methods.

**Figure 6.** B-mode US images of the left ventricle 4 h after conorary occlusion overlayed with the signal of E-selectin targeted MB (**A**), non-targeted MB (**B**) and non-specific IgG targeted MB (**C**). Enhanced contrast signal is seen using E-selectin targeted MB. Reproduced with permission from [88]. Copyright SAGE Publications, 2014.

Inflammatory bowel disease (IBD) is currently assessed by endoscopic monitoring, US, and clinical chemistry. Endoscopy is invasive and cannot access major parts of the small intestine. US is applied to capture the enhanced thickness of the inflamed bowel wall and the enhanced perfusion by Doppler. However, this approach has limited sensitivity and only becomes prominent at advanced disease stages. Here, molecular US could be introduced as a complementary sensitive tool. Inflammation in patients with IBD is associated with increased expression of cell adhesion molecules. Initially, mucosal addressin cell adhesion molecule 1 (MAdCAM-1) targeted MB were proposed for IBD detection [60]. The CEUS signal using these MB positively correlated with the severity of ileal inflammation assessed by immunohistochemical analysis. However, no further research was published using these MB. Instead, molecular US of IBD was mostly approached using different selectin-targeted MB. For example, P-selectin-targeted MB were successfully applied for IBD detection and monitoring of anti-TNFα antibody therapy [81]. CEUS signal changes were observed already after 3 days of therapy, while there was no decrease in bowel wall thickness or perfusion yet. In another study, these MB were able to depict radiation-induced P-selectin expression in the colon [82]. Furthermore, clinical-scale Pand E-selectin-targeted UCA were tested for IBD detection. The experiments in colitis induced mice showed that molecular US assessment of IBD excellently correlated with 18FDG-PET (positron emission tomography) and histological examination [130]. Further studies in swine demonstrated that dual-targeted UCA have the potential for clinical translation [131,132]. Wang and co-workers showed that, one hour after inflammation induction by exposing ileum to 2, 4, 6-trinitrobenzene

sulfonic acid (TNBS) and ethanol, a significant increase in CEUS signal occurred [131]. The US signal changed in line with the increased selectin expression and further increased with the progression of the inflammation. Moreover, P- and E-selectin-targeted MB also proved promising for long-term monitoring of anti-inflammatory IBD treatment in swine [132]. The combination treatment of prednisone and meloxicam reduced inflammation and downregulated selectin expression in the inflamed vasculature in the bowel. This molecular change was clearly depicted by molecular US imaging. Interestingly, there was no difference in the inflammation score obtained from the histological analysis between treated and control groups. This underlines the high sensitivity of selectin-targeted US imaging for IBD assessment and highlights its potential for clinical translation.
