Molecular Imaging Of Vulnerable Plaques In Rabbits Using Contrast-enhanced Ultrasound Targeting And Effects Of Chemokine Receptor CXCR4 In Atherosclerosis

Backgrounds and objectives:Atherosclerosis is the major diseases affecting human health, the vast majority of patients died of acute cardiovascular events without any premonitory symptoms. In recent years, the study found the main reason leading to acute cardiovascular events due to atherosclerotic plaque rupture and thrombosis, which depending on the vulnerability of plaque. Rupture of vulnerable plaques is the main cause of acute coronary syndrome and myocardial infarction. Such vulnerable plaque is characterized by a thin fibrous cap, a large lipid core, and an inflammatory infiltrate. Neovascularization density and the distribution are also a necessary factor for the growth and stability of atherosclerotic plaque. The neovascularization is an important pathology characteristic for atherosclerosis plaque. The role of neovascularization in plaque progression has previously been described and shown to be increased in the patients with acute coronary syndromes. Targeted imaging with ultrasound has great potential as a tool for elucidating the molecular mechanisms responsible for disease. It has important role in detecting plaque neovascularization, recognition plaque stability, monitoring clinical efficacy, predicting the occurrence of cardiovascular and cerebrovascular accident and early prevention through the application of targeted imaging.Biological effects of VEGF are mainly mediated by two tyrosin kinase receptors: VEGFR1 (Flt-1) and VEGFR2 (?k-1/KDR). VEGF receptor-2 (kinase insert domain containing receptor, KDR) is essential for vascular endothelial cell proliferation, migration, and promoting endothelial cell survival, increasinged vascular permeability and so on. Our aim was to develop a method for quantitative assessment of plaque neovascularization and an index of plaque vulnerability. Microbubbles targeting to VEGFR-2 were prepared by conjugation of biotinylated microbubbles which biotinylated VEGFR-2 antibody via Streptavidin. We use high-frequency ultrasound imaging to investigate whether molecular imaging with targeted UCA could be used for diagnosis of vulnerable plaques and evaluate the relationship between the video intensity of VEGFR-2 targeted UCA and the VEGFR-2 expression in vivo. Preliminary study a new vascular targeting enhanced ultrasound imaging of molecular imaging techniques for detection vulnerable plaque.Studies have confirmed that plaque angiogenesis in atherosclerosis formation and development plays an important role. Neovascularization in atherosclerosis are in the majority of immaturity, lack of pericytes and smooth muscle cells, larger permeability, conduciving to infiltration of macrophages into the plaque, aggregation within the plaque, promotion of inflammatory reaction, increasing plaque instability. CXC chemokine receptor 4 has been shown to play a critical role in chemotaxis and homing. There is also increasing evidence that this receptor links to angiogenesis. Vascular endothelial growth factor (VEGF), the main agent responsible for angiogenesis and vascular permeability, is a 45 Kda glycoprotein secreted in the vascular wall by endothelial and smooth muscle cells. The relationship between VEGF and CXCR4 in rabbit model of atherosclerotic plaques has been reported rarely. Ours study established atherosclerosis model to examine CXCR4 protein expression and VEGF mRNA expression at different time points and its correlation. To explore the effects of chemokine receptor CXCR4 in atherosclerosis angiogenesis and its possible mechanism.Methods:1. To investigate the GL-7 targeted contrast agent binding to abdominal aorta intima in vivo, and in vitro.Microbubbles targeting to GL-7 were prepared via electrostatic adsorption. Abdominal aorta intima injury model was created by high cholesterol diet. Twelve weeks later,ultrasound contrast imaging were performed with the targeted and the non-targeted contrast agent for the in vivo evaluation, respectively. The fluorescence staining in abdominal aorta intima was observed after dropping two kinds of contrast agents and the fluorescence intensity was analysised statistically.2. We investigate the relationship between the video intensity of vascular endothelial growth factor receptor 2 (VEGFR-2) targeted UCA and the VEGFR-2 expression in vulnerable plaques model in rabbits.Microbubbles targeting to VEGFR-2 were prepared by conjugation of biotinylated microbubbles which biotinylated VEGFR-2 antibody via Streptavidin (SA). Vulnerability was created by delivering recombinant p53 adenovirus to atherosclerotic plaques, which were established by high cholesterol diet and balloon endothelial injury. Twelve weeks later,the average video intensity of pre- and post-contrast ultrasound images was measured.VEGFR-2 expression and vascular density were quantified by immunohistochemical staining.3. Characterize the relationship between vascular endothelial growth factor receptor (VEGF) and chemokine receptor 4 (CXCR4) in rabbit model of atherosclerotic plaques .To explore the effects of chemokine receptor CXCR4 in atherosclerosis angiogenesis and its possible mechanism.The rabbit model of atherosclerotic plaques was established through aortic balloon injury and high cholesterol diet. Levels of CXCR4 protein and VEGF gene were detected by western blotting and fluorescent quantitative PCR, respectively at 4,8,12 weeks and the results were analyzed statistically. Immunohistochemical of atherosclerotic plaque detected CXCR4, CD34 expression and localization.Results:1. The peak video intensity of targeted group in injuried intima was higher than that of the non-targeted group (137. 49±13. 56 dB vs 111. 24±15. 23; P < 0.05). Aorta intima there was green fluorescence in aorta intima in targeted group while only weak green fluorescence in non-targeted group. The fluorescence intensity of the two groups has statistically significant difference on basis of the results of frozen section (76.74±13.64 vs31.17±14.81; P < 0.05).2. VEGFR-2 targeted microbubbles were preparated by Streptavidin. Abdominal aorta vulnerable plaque model has the typical pathological features of vulnerable plaque. Video intensity of VEGFR-2 targeted UCA in plaques was higher than that of the non-targeted UCA (144.42±17.70dB vs 106.88±9.38 dB; Z= -3.984, P=0.000). VEGFR2 expression was correlate with video intensity of targeted UCA (r2= 0.78, P=0.001), while there was no correlation in control group (r2=0.17, P=0.301). 3. At the three time points, the level of CXCR4 protein was 0.46±0.03, 0.58±0.05, 0.65±0.07, and the level of VEGFmRNA was 1.04±0.24,1.99±0.21, 3.97±0.75,respectively. Level of CXCR4 positively correlated with the level of VEGF (r2=0.869, P<0.05). CXCR4、CD34 immunohistochemical staining in the vascular intima and plaque can be seen within the many positive staining.Conclusions:1. GL-7 targeted microbubble contrast agent binds to arterial intima both in vivo, and in vitro. It can significantly enhance imaging of rabbit abdominal aorta intima.2. The magnitude of the ultrasonographic signal from the retained VEGFR-2 targeted UCA correlates with VEGFR-2 expression. These results validate the use of targeted UCA for ultrasound imaging of vulnerable abdominal artery in rabbits.3. The chemokine receptor CXCR4 and VEGF expression was positively correlated . The chemokine receptor CXCR4 in the plaque plays an important role in angiogenesis, and may contribute to plaque progress.