title: Detection and quantification of angiogenesis in experimental valve disease with integrin-targeted nanoparticles and 19-fluorine MRI/MRS.
abstract: 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 alphanubeta3 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 alphanubeta3 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 19F MRS was performed at 11.7T. Integrated 19F 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 alphanubeta3 integrin targeted PFC nanoparticles, 19F 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 alphanubeta3 integrin staining revealed the presence of neovasculature within the valve leaflets.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Medicine, Washington University School of Medicine, St Louis, MO, USA. eaw1@cec.wustl.edu
authors.forename: Emily A
authors.initials: EA
authors.lastname: Waters
authors.forename: Junjie
authors.initials: J
authors.lastname: Chen
authors.forename: John S
authors.initials: JS
authors.lastname: Allen
authors.forename: Huiying
authors.initials: H
authors.lastname: Zhang
authors.forename: Gregory M
authors.initials: GM
authors.lastname: Lanza
authors.forename: Samuel A
authors.initials: SA
authors.lastname: Wickline
mesh_terms: D000818:Animals; D001021:Aortic Valve / Q000378:metabolism / Q000473:pathology*; D001024:Aortic Valve Stenosis / Q000378:metabolism / Q000473:pathology*; D001665:Binding Sites; D001667:Binding, Competitive; D004195:Disease Models, Animal; D005461:Fluorine; D005466:Fluorocarbons / Q000378:metabolism*; D007150:Immunohistochemistry; D039302:Integrin alphaVbeta3 / Q000378:metabolism*; D008279:Magnetic Resonance Imaging; D053758:Nanoparticles; D009389:Neovascularization, Pathologic / Q000378:metabolism / Q000473:pathology*; D011817:Rabbits; D012598:Sclerosis; D015854:Up-Regulation
chemical_list: D005466:Fluorocarbons; D039302:Integrin alphaVbeta3; D005461:Fluorine