Detection and quantification of angiogenesis in experimental valve disease with integrin-targeted nanoparticles and 19-fluorine MRI/MRS

BACKGROUND: Angiogenesis is a critical early feature of atherosclerotic plaque development and may also feature prominently in the pathogenesis of aortic valve stenosis. It has been shown that MRI can detect and quantify specific molecules of interest expressed in cardiovascular disease and cancer by measuring the unique fluorine signature of appropriately targeted perfluorocarbon (PFC) nanoparticles. In this study, we demonstrated specific binding of α(ν)β(3 )integrin targeted nanoparticles to neovasculature in a rabbit model of aortic valve disease. We also showed that fluorine MRI could be used to detect and quantify the development of neovasculature in the excised aortic valve leaflets. METHODS: New Zealand White rabbits consumed a cholesterol diet for ~180 days and developed aortic valve thickening, inflammation, and angiogenesis mimicking early human aortic valve disease. Rabbits (n = 7) were treated with α(ν)β(3 )integrin targeted PFC nanoparticles or control untargeted PFC nanoparticles (n = 6). Competitive inhibition in vivo of nanoparticle binding (n = 4) was tested by pretreatment with targeted nonfluorinated nanoparticles followed 2 hours later by targeted PFC nanoparticles. 2 hours after treatment, aortic valves were excised and (19)F MRS was performed at 11.7T. Integrated (19)F spectral peaks were compared using a one-way ANOVA and Hsu's MCB (multiple comparisons with the best) post hoc t test. In 3 additional rabbits treated with α(ν)β(3 )integrin targeted PFC nanoparticles, (19)F spectroscopy was performed on a 3.0T clinical scanner. The presence of angiogenesis was confirmed by immunohistochemistry. RESULTS: Valves of rabbits treated with targeted PFC nanoparticles had 220% more fluorine signal than valves of rabbits treated with untargeted PFC nanoparticles (p < 0.001). Pretreatment of rabbits with targeted oil-based nonsignaling nanoparticles reduced the fluorine signal by 42% due to competitive inhibition, to a level not significantly different from control animals. Nanoparticles were successfully detected in all samples scanned at 3.0T. PECAM endothelial staining and α(ν)β(3 )integrin staining revealed the presence of neovasculature within the valve leaflets. CONCLUSION: Integrin-targeted PFC nanoparticles specifically detect early angiogenesis in sclerotic aortic valves of cholesterol fed rabbits. These techniques may be useful for assessing atherosclerotic components of preclinical aortic valve disease in patients and could assist in defining efficacy of medical therapies.